Method of treating cancer with composition comprising IL-15-based molecules and BCG

ABSTRACT

The invention features combination therapies using an IL-15-based superagonist complex and an antibody to effectively treat subjects with cancer and infectious diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/825,959, filed May 26, 2022, now U.S. Pat. No. 11,679,144, which is acontinuation of U.S. application Ser. No. 16/925,138, filed Jul. 9,2020, now U.S. Pat. No. 11,471,511, which is a continuation of U.S.application Ser. No. 16/444,807 filed Jun. 18, 2019, now U.S. Pat. No.11,173,191, which is a continuation of U.S. application Ser. No.15/921,512 filed Mar. 14, 2018, now U.S. Pat. No. 10,537,615, which is adivision of U.S. application Ser. No. 14/755,989 filed Jun. 30, 2015,now U.S. Pat. No. 9,925,247 which claims the benefit of priority under35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/018,899, filedJun. 30, 2014. The entire disclosures of each of which are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ST.26 XML format and is hereby incorporatedby reference in its entirety. Said XML copy, created on Dec. 9, 2022, isnamed PAT_000727_SL.xml and is 9000 bytes in size.

FIELD OF THE INVENTION

This invention relates generally to the field of therapies for treatmentof cancer and infectious agents.

BACKGROUND OF THE INVENTION

Prior to the invention described herein, there was a pressing need todevelop new strategies to augment and/or direct immune activity againstcancer and infected cells.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the surprising discoverythat an antibody in combination with ALT-803, a complex of aninterleukin-15 (IL-15) superagonist mutant and a dimeric IL-15 receptorα/Fc fusion protein, is useful for enhancing an immune response againsta neoplasia (e.g., a glioblastoma, prostate cancer, hematologicalcancer, B-cell neoplasms, multiple myeloma, B-cell lymphoma, Hodgkin'slymphoma, acute myeloid leukemia, chronic lymphocytic leukemia,cutaneous T-cell lymphoma, T-cell lymphoma, a solid tumor,urothelial/bladder carcinoma, melanoma, lung cancer, renal cellcarcinoma, breast cancer, gastric and esophageal cancer, head and neckcancer, colorectal cancer, ovarian cancer, non-small cell lungcarcinoma, B cell non-Hodgkin lymphoma, and squamous cell head and neckcarcinoma) or an infection (e.g., a viral infection with humanimmunodeficiency virus).

Methods for treating a neoplasia or an infection in a subject arecarried out by administering to the subject an effective amount of anantibody (or antibody-like molecule) and an effective amount of apharmaceutical composition comprising an IL-15N72D:IL-15RαSu/Fc complex(ALT-803), wherein the ALT-803 comprises a dimeric IL-15RαSu/Fc and twoIL-15N72D molecules. In one aspect, the IL-15N72D molecule comprises SEQID NO: 3. An exemplary IL-15RαSu/Fc comprises SEQ ID NO: 6.

The subject is preferably a mammal in need of such treatment, e.g., asubject that has been diagnosed with a neoplasia or an infection or apredisposition thereto. The mammal is any mammal, e.g., a human, aprimate, a mouse, a rat, a dog, a cat, a horse, as well as livestock oranimals grown for food consumption, e.g., cattle, sheep, pigs, chickens,and goats. In a preferred embodiment, the mammal is a human.

Suitable neoplasias for treatment with the methods described hereininclude a glioblastoma, prostate cancer, acute myeloid leukiemia, B-cellneoplasm, multiple myeloma, B-cell lymphoma, non-Hodgkin's lymphoma,chronic lymphocytic leukemia, cutaneous T-cell lymphoma, T-celllymphoma, a solid tumor, urothelial/bladder carcinoma, melanoma, lungcancer, renal cell carcinoma, breast cancer, gastric and esophagealcancer, head and neck cancer, colorectal cancer, and ovarian cancer. Anexemplary infection for treatment using the methods described herein isinfection with human immunodeficiency virus (HIV). The methods describedherein are also useful to treat bacterial infections (e.g., grampositive or gram negative bacteria) (Oleksiewicz et al. 2012. ArchBiochem Biophys. 526:124-31).

Preferably, administration of the compositions described herein alsoprevents future recurrence of neoplasia or infection after treatment ofthe disease.

Additionally, the methods of the invention are useful for effectivetreatment of autoimmune diseases, in which inhibition or reduction ofcells associated with the autoimmune responses provides clinical benefitto patients. Such cells include leucocytes, particularly B- or T-cellsand such autoimmune diseases include rheumatoid arthritis, juvenileidiopathic arthritis, psoriasis, Crohn's disease, ulcerative colitis,multiple sclerosis, ankylosing spondylitis, type 1 diabetes and systemiclupus erythematosus (Chan et al. 2010. Nat Rev Immunol. 10:301-16).

Exemplary effective doses of ALT-803 include between 0.1 μg/kg and 100mg/kg body weight, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, or 900μg/kg body weight or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, or 100 mg/kg body weight.

In some cases, the ALT-803 is administered daily, e.g., every 24 hours.Or, the ALT-803 is administered continuously or several times per day,e.g., every 1 hour, every 2 hours, every 3 hours, every 4 hours, every 5hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every10 hours, every 11 hours, or every 12 hours.

Exemplary effective daily doses of ALT-803 include between 0.1 μg/kg and100 μg/kg body weight, e.g., 0.1, 0.3, 0.5, 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 μg/kg bodyweight.

Alternatively, the ALT-803 is administered about once per week, e.g.,about once every 7 days. Or, the ALT-803 is administered twice per week,three times per week, four times per week, five times per week, sixtimes per week, or seven times per week. Exemplary effective weeklydoses of ALT-803 include between 0.0001 mg/kg and 4 mg/kg body weight,e.g., 0.001, 0.003, 0.005, 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, or 4mg/kg body weight. For example, an effective weekly dose of ALT-803 isbetween 0.1 μg/kg body weight and 400 μg/kg body weight. Alternatively,ALT-803 is administered at a fixed dose or based on body surface area(i.e., per m²).

In some cases, subjects receive two 6-week cycles consisting of 4 weeklyALT-803 intravenous doses followed by a 2-week rest period. Ultimately,the attending physician or veterinarian decides the appropriate amountand dosage regimen.

The compositions described herein are administered systemically,intravenously, subcutaneously, intramuscularly, intravesically, or byinstillation. The antibody and ALT-803 may be administeredsimultaneously or sequentially.

Preferably, the antibody (Ab) is a tumor-specific antibody, an immunecheckpoint inhibitor, or an antiviral antibody. Preferred antibodies arecomposed of heavy and light chain immunoglobulin (Ig) proteins, whichmay include rodent, human, chimeric and humanized forms. Additionally,the method described herein could utilize antibody-like molecules, suchas molecules comprising an antigen binding domain (e.g., single chainantibody, Fab, Fv, T-cell receptor binding domain, ligand binding domainor receptor binding domain). In some cases, these domains are preferablylinked to an Fc domain. The Ig may be of any of the known isotypes(e.g., IgA, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgG2a, IgG2b, andIgM). In some applications described herein using diseased targeted Abs(or antibody-like molecules), the Ab (or antibody-like molecule)contains a heavy chain or Fc domain capable of interacting with Fcreceptors to mediate antibody-dependent cell-mediated cytotoxicity(ADCC) and/or antibody dependent cellular phagocytosis (ADCP). In othercases, antibodies conjugated to effector molecules may be used. In otherapplications such use of immune checkpoint blocker, the preferred Ab (orantibody-like molecule) contains a heavy chain or Fc domain (e.g., IgG4Fc) that is incapable of effectively mediating ADCC or ADCP.

In certain embodiments, the antigen for the antibody comprises a cellsurface receptor or ligand. In a further embodiment, the antigencomprises a CD antigen, cytokine or chemokine receptor or ligand, growthfactor receptor or ligand, tissue factor, cell adhesion molecule,MHC/MHC-like molecules, Fc receptor, Toll-like receptor, NK receptor,TCR, BCR, positive/negative co-stimulatory receptor or ligand, deathreceptor or ligand, tumor associated antigen, or virus encoded antigen.

Preferably, the tumor-specific antibody is capable of binding to anantigen on a tumor cell. Tumor-specific antibodies approved fortreatment of patients with cancer include rituximab, ofatumumab, andobinutuzumab (anti-CD20 Abs); trastuzumab and pertuzumab (anti-HER2Abs); cetuximab and panitumumab (anti-EGFR Abs); and alemtuzumab(anti-CD52 Ab). Similarly, antibody-effector molecule conjugatesspecific to CD20 (⁹⁰Y-labeled ibritumomab tiuxetan, ¹³¹I-labeledtositumomab), HER2 (ado-trastuzumab emtansine), CD30 (brentuximabvedotin) and CD33 (gemtuzumab ozogamicin) have been approved for cancertherapy (Sliwkowski M X, Mellman I. 2013 Science 341:1192).

Additionally, preferred antibodies of the invention may include variousother tumor-specific antibodies known in the art. The antibodies andtheir respective targets for treatment of cancer include but are notlimited to nivolumab (anti-PD-1 Ab), TA99 (anti-gp75), 3F8 (anti-GD2),8H9 (anti-B7-H3), abagovomab (anti-CA-125 (imitation)), adecatumumab(anti-EpCAM), afutuzumab (anti-CD20), alacizumab pegol (anti-VEGFR2),altumomab pentetate (anti-CEA), amatuximab (anti-mesothelin), AME-133(anti-CD20), anatumomab mafenatox (anti-TAG-72), apolizumab(anti-HLA-DR), arcitumomab (anti-CEA), bavituximab(anti-phosphatidylserine), bectumomab (anti-CD22), belimumab(anti-BAFF), besilesomab (anti-CEA-related antigen), bevacizumab(anti-VEGF-A), bivatuzumab mertansine (anti-CD44 v6), blinatumomab(anti-CD19), BMS-663513 (anti-CD137), brentuximab vedotin (anti-CD30(TNFRSF8)), cantuzumab mertansine (anti-mucin CanAg), cantuzumabravtansine (anti-MUC1), capromab pendetide (anti-prostatic carcinomacells), carlumab (anti-MCP-1), catumaxomab (anti-EpCAM, CD3),cBR96-doxorubicin immunoconjugate (anti-Lewis-Y antigen), CC49(anti-TAG-72), cedelizumab (anti-CD4), Ch.14.18 (anti-GD2), ch-TNT(anti-DNA associated antigens), citatuzumab bogatox (anti-EpCAM),cixutumumab (anti-IGF-1 receptor), clivatuzumab tetraxetan (anti-MUC1),conatumumab (anti-TRAIL-R2), CP-870893 (anti-CD40), dacetuzumab(anti-CD40), daclizumab (anti-CD25), dalotuzumab (anti-insulin-likegrowth factor I receptor), daratumumab (anti-CD38 (cyclic ADP ribosehydrolase)), demcizumab (anti-DLL4), detumomab (anti-B-lymphoma cell),drozitumab (anti-DR5), duligotumab (anti-HER3), dusigitumab(anti-ILGF2), ecromeximab (anti-GD3 ganglioside), edrecolomab(anti-EpCAM), elotuzumab (anti-SLAMF7), elsilimomab (anti-IL-6),enavatuzumab (anti-TWEAK receptor), enoticumab (anti-DLL4), ensituximab(anti-SAC), epitumomab cituxetan (anti-episialin), epratuzumab(anti-CD22), ertumaxomab (anti-HER2/neu, CD3), etaracizumab(anti-integrin αvβ), faralimomab (anti-Interferon receptor),farletuzumab (anti-folate receptor 1), FBTA05 (anti-CD20), ficlatuzumab(anti-HGF), figitumumab (anti-IGF-1 receptor), flanvotumab (anti-TYRP1(glycoprotein 75)), fresolimumab (anti-TGF β), futuximab (anti-EGFR),galiximab (anti-CD80), ganitumab (anti-IGF-I), gemtuzumab ozogamicin(anti-CD33), girentuximab (anti-carbonic anhydrase 9 (CA-IX)),glembatumumab vedotin (anti-GPNMB), guselkumab (anti-IL13), ibalizumab(anti-CD4), ibritumomab tiuxetan (anti-CD20), icrucumab (anti-VEGFR-1),igovomab (anti-CA-125), IMAB362 (anti-CLDN18.2), IMC-CS4 (anti-CSF1R),IMC-TR1 (TGFβRII), imgatuzumab (anti-EGFR), inclacumab (anti-selectinP), indatuximab ravtansine (anti-SDC1), inotuzumab ozogamicin(anti-CD22), intetumumab (anti-CD51), ipilimumab (anti-CD152),iratumumab (anti-CD30 (TNFRSF8)), KM3065 (anti-CD20), KW-0761(anti-CD194), LY2875358 (anti-MET) labetuzumab (anti-CEA), lambrolizumab(anti-PDCD1), lexatumumab (anti-TRAIL-R2), lintuzumab (anti-CD33),lirilumab (anti-KIR2D), lorvotuzumab mertansine (anti-CD56), lucatumumab(anti-CD40), lumiliximab (anti-CD23 (IgE receptor)), mapatumumab(anti-TRAIL-R1), margetuximab (anti-ch4D5), matuzumab (anti-EGFR),mavrilimumab (anti-GMCSF receptor α-chain), milatuzumab (anti-CD74),minretumomab (anti-TAG-72), mitumomab (anti-GD3 ganglioside),mogamulizumab (anti-CCR4), moxetumomab pasudotox (anti-CD22), nacolomabtafenatox (anti-C242 antigen), naptumomab estafenatox (anti-5T4),narnatumab (anti-RON), necitumumab (anti-EGFR), nesvacumab(anti-angiopoietin 2), nimotuzumab (anti-EGFR), nivolumab (anti-IgG4),nofetumomab merpentan, ocrelizumab (anti-CD20), ocaratuzumab(anti-CD20), olaratumab (anti-PDGF-R a), onartuzumab (anti-c-MET),ontuxizumab (anti-TEM1), oportuzumab monatox (anti-EpCAM), oregovomab(anti-CA-125), otlertuzumab (anti-CD37), pankomab (anti-tumor specificglycosylation of MUC1), parsatuzumab (anti-EGFL7), pascolizumab(anti-IL-4), patritumab (anti-HER3), pemtumomab (anti-MUC1), pertuzumab(anti-HER2/neu), pidilizumab (anti-PD-1), pinatuzumab vedotin(anti-CD22), pintumomab (anti-adenocarcinoma antigen), polatuzumabvedotin (anti-CD79B), pritumumab (anti-vimentin), PRO131921 (anti-CD20),quilizumab (anti-IGHE), racotumomab (anti-N-glycolylneuraminic acid),radretumab (anti-fibronectin extra domain-B), ramucirumab (anti-VEGFR2),rilotumumab (anti-HGF), robatumumab (anti-IGF-1 receptor), roledumab(anti-RHD), rovelizumab (anti-CD11 & CD18), samalizumab (anti-CD200),satumomab pendetide (anti-TAG-72), seribantumab (anti-ERBB3), SGN-CD19A(anti-CD19), SGN-CD33A (anti-CD33), sibrotuzumab (anti-FAP), siltuximab(anti-IL-6), solitomab (anti-EpCAM), sontuzumab (anti-episialin),tabalumab (anti-BAFF), tacatuzumab tetraxetan (anti-alpha-fetoprotein),taplitumomab paptox (anti-CD19), telimomab aritox, tenatumomab(anti-tenascin C), teneliximab (anti-CD40), teprotumumab (anti-CD221),TGN1412 (anti-CD28), ticilimumab (anti-CTLA-4), tigatuzumab(anti-TRAIL-R2), TNX-650 (anti-IL-13), tositumomab (anti-CS20),tovetumab (anti-CD140a), TRBS07 (anti-GD2), tregalizumab (anti-CD4),tremelimumab (anti-CTLA-4), TRU-016 (anti-CD37), tucotuzumab celmoleukin(anti-EpCAM), ublituximab (anti-CD20), urelumab (anti-4-1BB),vantictumab (anti-Frizzled receptor), vapaliximab (anti-AOC3 (VAP-1)),vatelizumab (anti-ITGA2), veltuzumab (anti-CD20), vesencumab(anti-NRP1), visilizumab (anti-CD3), volociximab (anti-integrin α5β1),vorsetuzumab mafodotin (anti-CD70), votumumab (anti-tumor antigenCTAA16.88), zalutumumab (anti-EGFR), zanolimumab (anti-CD4), zatuximab(anti-HER1), ziralimumab (anti-CD147 (basigin)), RG7636 (anti-ETBR),RG7458 (anti-MUC16), RG7599 (anti-NaPi2b), MPDL3280A (anti-PD-L1),RG7450 (anti-STEAP1), and GDC-0199 (anti-Bcl-2).

Other antibodies or tumor target binding proteins useful in theinvention (e.g. TCR domains) include, but are not limited to, those thatbind the following antigens (the cancer indications representnon-limiting examples): aminopeptidase N (CD13), annexin A1, B7-H3(CD276, various cancers), CA125 (ovarian cancers), CA15-3 (carcinomas),CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X(carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectalcancers), placental alkaline phosphatase (carcinomas), prostate specificantigen (prostate), prostatic acid phosphatase (prostate), epidermalgrowth factor (carcinomas), CD2 (Hodgkin's disease, NHL lymphoma,multiple myeloma), CD3 epsilon (T cell lymphoma, lung, breast, gastric,ovarian cancers, autoimmune diseases, malignant ascites), CD19 (B cellmalignancies), CD20 (non-Hodgkin's lymphoma, B-cell neoplasmas,autoimmune diseases), CD21 (B-cell lymphoma), CD22 (leukemia, lymphoma,multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia,autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiplemyeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52(leukemia), CD56 (small cell lung cancers, ovarian cancer, Merkel cellcarcinoma, and the liquid tumor, multiple myeloma), CD66e (carcinomas),CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74(multiple myeloma), CD80 (lymphoma), CD98 (carcinomas), CD123(leukemia), mucin (carcinomas), CD221 (solid tumors), CD227 (breast,ovarian cancers), CD262 (NSCLC and other cancers), CD309 (ovariancancers), CD326 (solid tumors), CEACAM3 (colorectal, gastric cancers),CEACAM5 (CEA, CD66e) (breast, colorectal and lung cancers), DLL4(A-like-4), EGFR (various cancers), CTLA4 (melanoma), CXCR4 (CD 184,heme-oncology, solid tumors), Endoglin (CD 105, solid tumors), EPCAM(epithelial cell adhesion molecule, bladder, head, neck, colon, NHLprostate, and ovarian cancers), ERBB2 (lung, breast, prostate cancers),FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancers),FGFR (carcinomas), GD2 ganglioside (carcinomas), G-28 (a cell surfaceantigen glycolipid, melanoma), GD3 idiotype (carcinomas), heat shockproteins (carcinomas), HER1 (lung, stomach cancers), HER2 (breast, lungand ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia),human chorionic gonadotropin (carcinomas), IGF1R (solid tumors, bloodcancers), IL-2 receptor (T-cell leukemia and lymphomas), IL-6R (multiplemyeloma, RA, Castleman's disease, IL6 dependent tumors), integrins(αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, for various cancers), MAGE-1(carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4(carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma),MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-Hodgkin's Bcell lymphoma, leukemia), MUC1 (breast, ovarian, cervix, bronchus andgastrointestinal cancer), MUC16 (CA125) (ovarian cancers), CEA(colorectal cancer), gp100 (melanoma), MARTI (melanoma), MPG (melanoma),MS4A1 (membrane-spanning 4-domains subfamily A, small cell lung cancers,NHL), nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas),nectin-4 (carcinomas), paratope of anti-(N-glycolylneuraminic acid,breast, melanoma cancers), PLAP-like testicular alkaline phosphatase(ovarian, testicular cancers), PSMA (prostate tumors), PSA (prostate),ROB04, TAG 72 (tumour associated glycoprotein 72, AML, gastric,colorectal, ovarian cancers), T cell transmembrane protein (cancers),Tie (CD202b), tissue factor, TNFRSF10B (tumor necrosis factor receptorsuperfamily member 10B, carcinomas), TNFRSF13B (tumor necrosis factorreceptor superfamily member 13B, multiple myeloma, NHL, other cancers,RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma),TRAIL-R1 (tumor necrosis apoptosis inducing ligand receptor 1, lymphoma,NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma), VEGF, VEGF-A,VEGF-2 (CD309) (various cancers). Some other tumor associated antigentargets have been reviewed (Gerber, et al, mAbs 2009 1:247-253;Novellino et al, Cancer Immunol Immunother. 2005 54:187-207, Franke, etal, Cancer Biother Radiopharm. 2000, 15:459-76, Guo, et al., Adv CancerRes. 2013; 119: 421-475, Parmiani et al. J Immunol. 2007 178:1975-9).Examples of these antigens include Cluster of Differentiations (CD4,CD5, CD6, CD7, CD8, CD9, CD10, CD1 1a, CD1 1b, CD1 1c, CD12w, CD14,CD15, CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29,CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46,CD47, CD48, CD49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E,CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83,CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD100, CD103, CD105,CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, CD138, CD141,CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163,CD166, CD168, CD184, CDw186, CD195, CD202 (a, b), CD209, CD235a, CD271,CD303, CD304), annexin A1, nucleolin, endoglin (CD105), ROB04,amino-peptidase N, -like-4 (DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2,B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, idiotype, MAGEA3, p53 nonmutant, NY-ESO-1, GD2, CEA, MelanA/MART1, Ras mutant, gp100,p53 mutant, proteinase3 (PR1), bcr-abl, tyrosinase, survivin, hTERT,sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG(TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GMl, mesothelin,PSCA, MAGE A1, sLe(a), CYPIB I, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML,NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, spermprotein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2,Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, and Fos-related antigen1.

Additionally, preferred antibodies of the invention include thosespecific to antigens and epitope targets associated with infected cellsthat are known in the art. Such targets include but are not limitedthose derived from the following infectious agents are of interest: HIVvirus (particularly antigens derived from the HIV envelope spike and/orgp120 and gp41 epitopes), Human papilloma virus (HPV), Mycobacteriumtuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasmacapsulatum,—influenzae B, Treponema pallidum, Lyme disease spirochetes,Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabiesvirus, influenza virus, cytomegalovirus, herpes simplex virus I, herpessimplex virus II, human serum parvo-like virus, respiratory syncytialvirus, varicella-zoster virus, hepatitis B virus, hepatitis C virus,measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barrvirus, murine leukemia virus, mumps virus, vesicular stomatitis virus,sindbis virus, lymphocytic choriomeningitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubellavirus, West Nile virus, Plasmodium falciparum, Plasmodium vivax,Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosomarhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosomajaponicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus,Leishmania tropica, Trichinella spiralis, Theileria parva, Taeniahydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus,Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale,Marginini, Acholeplasma laidlawii, M. salivarium and M. pneumoniae.

In other embodiments, the antibody (or antibody-like molecule) isspecific to an immune checkpoint molecule or its ligand and acts as aninhibitor of immune checkpoint suppressive activity or as an agonist ofimmune checkpoint stimulatory activity. Such immune checkpoint moleculesand ligands include PD1, PDL1, PDL2, CTLA4, CD28, CD80, CD86, B7-H3,B7-H4, B7-H5, ICOS-L, ICOS, BTLA, CD137L, CD137, HVEM, KIR, 4-4BB,OX40L, CD70, CD27, OX40, GITR, IDO, TIM3, GAL9, VISTA, CD155, TIGIT,LIGHT, LAIR-1, Siglecs and A2aR (Pardoll D M. 2012. Nature Rev Cancer12:252-264, Thaventhiran T, et al. 2012. J Clin Cell Immunol S12:004).Additionally, preferred antibodies of the invention may includeipilimumab and tremelimumab (anti-CTLA4). nivolumab, pembrolizumab,pidilizumab, TSR-042, ANBO11, AMP-514 and AMP-224 (a ligand-Fc fusion)(anti-PD1), MPDL3280A, MEDI4736, MEDI0680, and BMS-9365569 (anti-PDL1),MEDI6469 (anti-OX40 agonist), BMS-986016, IMP701, IMP731, and IMP321(anti-LAG3).

In one aspect, the addition of ALT-803 to antibody treatment in vitro orin vivo increases cytotoxicity of immune cells against diseased ordisease-associated cells. In some cases, ALT-803 is capable ofstimulating immune cells to augment ADCC or ADCP activity against tumor,infected or autoimmune disease-associated cells mediated by adisease-specific antibody (or antibody-like molecule). In oneembodiment, treatment of immune cells with ALT-803 increases ADCC orADCP activity against diseased of disease-associated cells mediated by adisease target-specific antibody by at least 5%, e.g., at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%. In a preferred embodiment,immune cells are treated with ALT-803 and used to kill tumor cells viaADCC or ADCP mediated a tumor-specific antibody, wherein the level oftumor cell death is at least 5% greater, e.g., at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 100% greater than that seen with immunecells that were not treated with ALT-803. In preferred embodiments,tumor-specific ADCC or ADCP in the subject is augmented by at least 5%,e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 100%following the ALT-803 and antibody administration. In particularembodiments, NK cell-based ADCC activity is augmented by ALT-803treatment.

In other cases, ALT-803 stimulates CD4⁺ and CD8⁺ T cells to killdiseased or disease-associated cells, e.g., tumor cells or infectedcells. Preferably, ALT-803 treatment stimulates immune cell cytolyticactivity and immune checkpoint blockers treatment inhibitsimmunosuppressive responses, such that in combination these treatmentsprovide highly effective and/or durable activity against the tumor orinfected cells. In some embodiments, ALT-803 increases serum levels ofinterferon gamma (IFN-7) and/or IL-6, stimulates NK and T cellproliferation and upregulated NK and T cell expression of activationmarkers including CD25, CD69, perforin and granzyme. Induction of thesemarkers may enhance the responsiveness or cytolytic activity of theimmune cells against diseased cells. For example, the methods describedherein stimulate NK cells to kill tumor or infected cells.

In other embodiments, ALT-803 induces the activity and/or level of otherinnate immune cells including neutrophils or monocytic cells. Such cellsare known to mediate ADCC and ADCP of therapeutic antibodies againstdiseased cells, e.g., tumor cells or infected cells (Golay, et al.Blood. 2013 122:3482-91, Richards, et al, Mol Cancer Ther 20087:2517-27). Preferably, combination therapy of ALT-803 and antibodiesprovides improve clinical responses in patients with cancer orinfections through a mechanism that is mediated, at least in part, byinnate immune cells. For example, the methods described herein stimulateneutrophils or monocytic cells to kill tumor or infected cells.

Preferably, the methods described herein result in a reduced/decreasednumber of tumor or infected cells compared to the number of tumor orinfected cells prior to administration of the compositions herein.Alternatively, the methods described herein result in a decreaseddisease progression of the neoplasia or infection. Preferably, themethods described herein result in prolonged survival of a subjectcompared to untreated subjects.

In some cases, methods for treating a neoplasia or an infection in asubject are carried out by administering to the subject an effectiveamount of Bacillus Calmette-Guerin (BCG) and an effective amount of apharmaceutical composition comprising ALT-803, wherein the ALT-803comprises a dimeric IL-15RαSu/Fc and two IL-15N72D molecules. Forexample, subjects receive BCG plus ALT-803 weekly via a urinary catheterin the bladder for 6 consecutive weeks.

Also provided is a kit for the treatment of a neoplasia, the kitcomprising an effective amount of ALT-803, an antibody, and directionsfor the use of the kit for the treatment of a neoplasia.

A kit for the treatment of an infection comprises an effective amount ofALT-803, an antibody, and directions for the use of the kit for thetreatment of an infection.

In certain aspects of the soluble fusion protein complexes of theinvention, the IL-15 polypeptide is an IL-15 variant having a differentamino acid sequence than native IL-15 polypeptide. The human IL-15polypeptide is referred to herein as huIL-15, hIL-15, huIL15, hIL15,IL-15 wild type (wt), and variants thereof are referred to using thenative amino acid, its position in the mature sequence and the variantamino acid. For example, huIL15N72D refers to human IL-15 comprising asubstitution of N to D at position 72. In one aspect, the IL-15 variantfunctions as an IL-15 agonist as demonstrated, e.g., by increasedbinding activity for the IL-15RβγC receptors compared to the nativeIL-15 polypeptide. Alternatively, the IL-15 variant functions as anIL-15 antagonist as demonstrated by e.g., decreased binding activity forthe IL-15RβγC receptors compared to the native IL-15 polypeptide.

Methods for killing a target cell are carried out by contacting aplurality of cells with an antibody and ALT-803, wherein the pluralityof cells further include immune cells bearing the IL-15R chainsrecognized by the IL-15 domain, or immune cells bearing Fc receptorchains recognized by the Fc domain, and the target cells bearing anantigen recognized by an the antibody (e.g., an anti-CD20 antibody), andkilling the target cells. For example, the target cells are tumor cellsor infected (e.g., virally infected) cells.

A method for killing diseased cells expressing a target antigen iscarried out by treating immune cells with an effective amount of anIL-15N72D:IL-15RαSu/Fc complex (ALT-803), mixing the ALT-803-treatedimmune cells with an antibody specific to a target antigen and diseasedcells expressing said target antigen, and killing the diseased cells viaADCC or ADCP mediated by the ALT-803-treated immune cells and targetantigen-specific antibody. In one aspect, the level of diseased cellkilling is increased by at least 5%, e.g., at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100% compared to that mediated by immune cellsthat were not treated with ALT-803.

The invention also provides methods for preventing or treating diseasein a patient in which the diseased cells express a disease associatedantigen, the method including the steps of: contacting a plurality ofcells with an antibody and ALT-803, and damaging or killing the diseasecells sufficient to prevent or treat the disease in the patient. Inpreferred embodiments, combination therapy with ALT-803 and an antibodycan decrease disease progression and/or prolong patient survival.

The invention provides methods of stimulating immune responses in amammal by administering to the mammal an effective amount of an antibodyand an effective amount of ALT-803.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant a peptide, nucleic acid molecule, or small compound.An exemplary therapeutic agent is ALT-803.

By “ALT-803” is meant a complex comprising IL-15N72D noncovalentlyassociated with a dimeric IL-15RαSu/Fc fusion protein and having immunestimulating activity. In one embodiment, the IL-15N72D and/orIL-15RαSu/Fc fusion protein comprises one, two, three, four or moreamino acid variations relative to a reference sequence. An exemplaryIL-15N72D amino acid sequence is provided below.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

The invention includes antibodies or fragments of such antibodies, solong as they exhibit the desired biological activity. Also included inthe invention are chimeric antibodies, such as humanized antibodies.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. Humanization can beperformed, for example, using methods described in the art, bysubstituting at least a portion of a rodent complementarity-determiningregion for the corresponding regions of a human antibody.

The term “antibody” or “immunoglobulin” is intended to encompass bothpolyclonal and monoclonal antibodies. The preferred antibody is amonoclonal antibody reactive with the antigen. The term “antibody” isalso intended to encompass mixtures of more than one antibody reactivewith the antigen (e.g., a cocktail of different types of monoclonalantibodies reactive with the antigen). The term “antibody” is furtherintended to encompass whole antibodies, biologically functionalfragments thereof, single-chain antibodies, and genetically alteredantibodies such as chimeric antibodies comprising portions from morethan one species, bifunctional antibodies, antibody conjugates,humanized and human antibodies. Biologically functional antibodyfragments, which can also be used, are those peptide fragments derivedfrom an antibody that are sufficient for binding to the antigen.“Antibody” as used herein is meant to include the entire antibody aswell as any antibody fragments (e.g. F(ab′)2, Fab′, Fab, Fv) capable ofbinding the epitope, antigen or antigenic fragment of interest.

By “binding to” a molecule is meant having a physicochemical affinityfor that molecule.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include neoplasias and infections.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component, alone or in a combination, to providethe desired effect. For example, by “an effective amount” is meant anamount of a compound, alone or in a combination, required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. For example, a fragment maycontain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 nucleotides or amino acids. However, theinvention also comprises polypeptides and nucleic acid fragments, solong as they exhibit the desired biological activity of the full lengthpolypeptides and nucleic acid, respectively. A nucleic acid fragment ofalmost any length is employed. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length (including all intermediate lengths) are included inmany implementations of this invention. Similarly, a polypeptidefragment of almost any length is employed. For example, illustrativepolypeptide segments with total lengths of about 10,000, about 5,000,about 3,000, about 2,000, about 1,000, about 5,000, about 1,000, about500, about 200, about 100, or about 50 amino acids in length (includingall intermediate lengths) are included in many implementations of thisinvention.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation.

A “purified” or “biologically pure” protein is sufficiently free ofother materials such that any impurities do not materially affect thebiological properties of the protein or cause other adverseconsequences. That is, a nucleic acid or peptide of this invention ispurified if it is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Purity and homogeneity are typically determined using analyticalchemistry techniques, for example, polyacrylamide gel electrophoresis orhigh performance liquid chromatography. The term “purified” can denotethat a nucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. For a protein that can be subjected tomodifications, for example, phosphorylation or glycosylation, differentmodifications may give rise to different isolated proteins, which can beseparately purified.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

By “isolated nucleic acid” is meant a nucleic acid that is free of thegenes which flank it in the naturally-occurring genome of the organismfrom which the nucleic acid is derived. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones. Forexample, the isolated nucleic acid is a purified cDNA or RNApolynucleotide. Isolated nucleic acid molecules also include messengerribonucleic acid (mRNA) molecules.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “neoplasia” is meant a disease or disorder characterized by excessproliferation or reduced apoptosis. Illustrative neoplasms for which theinvention can be used include, but are not limited to leukemias (e.g.,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,acute myeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, gastric and esophagealcancer, head and neck cancer, rectal cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, glioblastomamultiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Inparticular embodiments, the neoplasia is multiple myeloma, beta-celllymphoma, urothelial/bladder carcinoma or melanoma. As used herein,“obtaining” as in “obtaining an agent” includes synthesizing,purchasing, or otherwise acquiring the agent.

By “reduces” is meant a negative alteration of at least 5%, 10%, 25%,50%, 75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout ortherebetween.

By “specifically binds” is meant a compound or antibody that recognizesand binds a polypeptide of the invention, but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule.Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100.mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferredembodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mMtrisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Usefulvariations on these conditions will be readily apparent to those skilledin the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.The subject is preferably a mammal in need of such treatment, e.g., asubject that has been diagnosed with B cell lymphoma or a predispositionthereto. The mammal is any mammal, e.g., a human, a primate, a mouse, arat, a dog, a cat, a horse, as well as livestock or animals grown forfood consumption, e.g., cattle, sheep, pigs, chickens, and goats. In apreferred embodiment, the mammal is a human.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. It will be appreciated that,although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated.

Treatment of patients with neoplasia may include any of the following:Adjuvant therapy (also called adjunct therapy or adjunctive therapy) todestroy residual tumor cells that may be present after the known tumoris removed by the initial therapy (e.g. surgery), thereby preventingpossible cancer reoccurrence; neoadjuvant therapy given prior to thesurgical procedure to shrink the cancer; induction therapy to cause aremission, typically for acute leukemia; consolidation therapy (alsocalled intensification therapy) given once a remission is achieved tosustain the remission; maintenance therapy given in lower or lessfrequent doses to assist in prolonging a remission; first line therapy(also called standard therapy); second (or 3rd, 4th, etc.) line therapy(also called salvage therapy) is given if a disease has not responded orreoccurred after first line therapy; and palliative therapy (also calledsupportive therapy) to address symptom management without expecting tosignificantly reduce the cancer.

The terms “preventing” and “prevention” refer to the administration ofan agent or composition to a clinically asymptomatic individual who issusceptible or predisposed to a particular adverse condition, disorder,or disease, and thus relates to the prevention of the occurrence ofsymptoms and/or their underlying cause.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1F are a series of line graphs demonstrating the effects ofALT-803 on in vitro proliferation of human immune cell subsets. HumanPBMCs were separated from blood buffy coats of two healthy donors(Donor-A, FIG. 1A, FIG. 1C, and FIG. 1E; Donor-B, FIG. 1B, FIG. 1D andFIG. 1F) and cultured in RPMI-10 with or without ALT-803 for 5 days(FIG. 1A and FIG. 1B) or the indicated times (FIG. 1C, FIG. 1D, FIG. 1Eand FIG. 1F). ALT-803 was added at the concentrations indicated in FIG.1A and FIG. 1B, and at 10 nM with RPMI-10 serving as a medium control(Ctrl) for the studies shown in FIGS. 1C-FIG. 1F. At the end of theincubation, PBMCs were stained with fluorochrome-labeled antibodiesspecific to CD4, CD8 (as markers for T cells) and CD16 (as a marker ofNK cells). The percentages of the cell subsets were analyzed on aFACSverse with FACSuite software. Triplicate samples from individualdonors were analyzed for FIG. 1A and FIG. 1B and single samples for eachtimepoint were analyzed for FIG. 1C and FIG. 1D. FIG. 1E and FIG. 1Frepresent the CD4/CD8 ratios determined from the results obtained inFIG. 1C and FIG. 1D, respectively.

FIG. 2A and FIG. 2B show dot plots demonstrating the effects of ALT-803on in vitro proliferation of human immune cell subsets from differentdonors. Human PBMCs were separated from blood buffy coats of 7 healthydonors and cultured in media alone or media containing 0.5 nM IL-15 orALT-803 for 7 days. At the end of the incubation, PBMCs were counted andstained with fluorochrome-labeled antibodies specific to immune cellsubsets. The percentages of the cell subsets were analyzed on aFACSverse with FACSuite software and the absolute cell counts werecalculated, as shown in FIG. 2A and FIG. 2B.

FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D are line graphs that demonstrateupregulation of CD25 and CD69 molecules on immune cell subsets followingincubation with ALT-803. Human PBMCs were separated from blood buffycoats of two healthy donors (left and right panels) and cultured inRPMI-10 with ALT-803 at the indicated concentration for 5 days. ALT-803activated PBMCs were stained with fluorochrome-labeled antibodiesspecific to CD4, CD8 (as markers of T cells), CD335 (as markers of NKcells), CD25 and CD69 (as activation markers). The fluorescent intensity[Geometric mean (MFI)] of CD25 and CD69 expression on CD4⁺ T cells, CD8⁺T cells and NK cells was analyzed on a FACSverse with FACSuite software.

FIG. 4A-FIG. 4D are line graphs that demonstrate upregulation ofgranzyme B and perforin expression in human CD8⁺ T cells and NK cells byALT-803. Human PBMCs were separated from blood buffy coats and culturedin RPMI-10 with ALT-803 at the indicated concentrations for 5 days.ALT-803-activated PBMCs were stained with fluorochrome-labeledantibodies specific to CD8, CD16 and CD335 (as markers of NK cells) andthen intracellularly stained with fluorochrome-labeled antibodiesspecific to granzyme B or perforin. The mean fluorescent intensity (MFI:Geometric mean) of granzyme B and perforin expression on CD8⁺ T cells(FIG. 4A: donor-1 and FIG. 4C: donor-2) and NK cells (FIG. 4B: donor-1and FIG. 4D: donor-2) was analyzed on a FACSverse with FACSuitesoftware.

FIG. 5A, FIG. 5B, and FIG. 5C are a series of bar charts demonstratingthe effects of ALT-803 on cytokine production and cell proliferation byhuman and mouse immune cells in culture. Human PBMCs (FIG. 5A) and mousesplenocytes (FIG. 5B) were incubated in media containing ALT-803 asindicated for 4 days. Changes in cytokines secreted in the cell culturemedia are shown in FIG. 5A and FIG. 5B. Changes in cell proliferativeresponses based on violet dye dilution are shown in FIG. 5C.

FIG. 6A-FIG. 6D are a series of line graphs and a bar chartdemonstrating cytotoxicity of human PBMCs against Daudi human B-celllymphoma and K562 human myelogenous leukemia cells induced by ALT-803.Human PBMCs were used as effector cells and Celltrace Violet-labeledDaudi and K562 cells were used as target cells. The human PBMCs weremixed with K562 cells (FIG. 6A), Daudi cells (FIG. 6B) or Daudi cellswith Rituximab (anti-CD20 Ab) at 10 nM (FIG. 6C) at the indicated E:Tratio in RPMI-10 with or without ALT-803 at 10 nM. The cell mixtureswere incubated at 37° C. for 3 days and the viability of Daudi and K562target cells was assessed by analysis of propidium iodide staining ofviolet-labeled target cells on a FACSVerse flow cytometer. Human PBMCswere mixed with violet-labeled K562 or Daudi cells at 10:1 ratio or withDaudi cells plus Rituximab (ADCC) at 2:1 ratio in RPMI-10 with orwithout ALT-803 at 10 nM. Following 1 to 3 days of incubation at 37° C.,the % cytotoxicity of the target cells was determined (FIG. 6D).

FIG. 7A and FIG. 7B are a series of line graphs demonstrating ALT-803concentration dependent induction of human PBMC cytotoxicity againstK562 and Daudi cells. Human PBMCs were used as effector cells andCelltrace Violet-labeled Daudi and K562 cells were used as target cells.Human PBMCs from two donors (A and B) were mixed with violet-labeledK562 cells or Daudi cells at E:T ratio of 20:1 in RPMI-10 with theindicated concentrations of ALT-803. Following a 3-day incubation at 37°C., the viability of Daudi and K562 target cells was assessed byanalysis of propidium iodide staining of violet-labeled target cells ona FACSVerse flow cytometer.

FIG. 8A-FIG. 8D are a series of line graphs showing that ALT-803augments ADCC of tumor specific Ab against tumor cells. Fresh humanPBMCs from donor-1 (FIG. 8A) or donor-2 (FIG. 8B) were mixed withviolet-labeled CD20-positive Daudi human B-cell lymphoma cells atE:T=2:1 in RPMI-10 with ALT-803 at the indicated concentration (0.01-10nM) alone or with Rituximab (anti-CD20 Ab) at 10 nM. After 2 days ofincubation at 37° C., the Daudi cell viability was assessed by analysisof propidium iodide staining of violet-labeled Daudi cells on a BDFACSVerse. In a follow-up study, NK cells were isolated from normalhuman PBMCs by MACS and used as effector cells. NK cells (FIG. 8C) andNK-depleted PBMCs (FIG. 8D) were mixed with violet-labeled Daudi cellsat E:T=1:1 in RPMI-10 containing ALT-803 at the indicated concentration(0.01 or 0.1 nM) with and without Rituximab or HOAT Ab at 10 nM. Thecells were incubated at 37° C. for 2 days. Daudi cell viability wasassessed by analysis of propidium iodide stained violet-labeled Daudicells on a BD FACSVerse.

FIG. 9A and FIG. 9B are a bar chart and a line graph showing thatALT-803 augments ADCC of tumor specific Ab against tumor cells by mousesplenocytes. In the study shown in FIG. 9A, splenocytes were isolatedfrom tumor-bearing SCID mice following treatment with PBS, ALT-803 (0.2mg/kg), Rituximab (10 mg/kg) or ALT-803+Rituximab. The splenocytes andCelltrace Violet-labeled Daudi cells were mixed at E:T=20:1 in thepresence of medium alone or medium containing ALT-803 (10 nM), Rituximab(10 nM) or ALT-803+Rituximab. After 2 days of incubation at 37° C. for 2days, Daudi target cell viability was assessed. Splenocytes were alsoisolated from Balb/c mice following treatment with ALT-803 (0.2 mg/kg)(FIG. 9B). The splenocytes and Celltrace Violet-labeled HER2-positiveSK-BR-3 human breast cancer cells were mixed at E:T=10:1 in the presenceof medium alone or medium containing various concentrations of anti-HER2antibody (clone 24D2), ALT-803 or both agents. After 24 hours ofincubation at 37° C., SK-BR-3 target cell viability was assessed.

FIG. 10 is a bar chart showing the antitumor efficacy of ALT-803 plusanti-CD20 antibody against human B lymphoma in SCID mice. Fox Chase SCIDfemale mice bearing Daudi cell tumors were treated with PBS, ALT-803(0.2 mg/kg), Rituximab (10 mg/kg) or ALT-803+Rituximab. Daudi cells inbone marrow were determined 4 days after the last treatment.

FIG. 11 is a bar graph showing the antitumor efficacy of ALT-803 plusanti-CD20 antibody against human B lymphoma in SCID mice. Fox Chase SCIDfemale mice bearing Daudi cell tumors were treated with PBS, Rituximabor Rituximab plus various concentrations of ALT-803. Daudi cells in bonemarrow were determined 4 days after the last treatment.

FIG. 12 is a line graph demonstrating prolonged survival of mice bearingDaudi cell tumors following ALT-803 plus anti-CD20 Ab therapy. Fox ChaseSCID female mice bearing Daudi cell tumors were treated with PBS,Rituximab (10 mg/kg), ALT-803 (0.05 mg/kg) or Rituximab plus ALT-803.The survival of the mice was monitored and Kaplan-Meier survival curveswere plotted.

FIG. 13A and FIG. 13B are a series of line graphs showing the prolongedsurvival of mice bearing CT26 colon carcinoma lung metastases followingALT-803 and ALT-803 plus anti-CTLA-4 Ab therapy. BALB/c mice bearingCT26 colon carcinoma lung metastases were treated with PBS, ALT-803,IL-15 monotherapies and combination therapies with anti-CTLA4 Ab andanti-PD-L1 Ab as indicated in the figures. The survival of the mice wasmonitored and Kaplan-Meier survival curves were plotted.

FIG. 14A and FIG. 14B are a series of line graphs demonstrating theprolonged survival of mice bearing 5T33P myeloma tumors followingALT-803 plus anti-PD-L1 Ab therapy. In FIG. 14A, C57BL6 mice bearing5T33P myeloma tumors were treated with PBS, ALT-803, IL-15 monotherapiesand combination therapies with anti-CTLA4 Ab and anti-PD-L1 Ab (0.2mg/mouse) as indicated in the figure. The survival of the mice wasmonitored and Kaplan-Meier survival curves were plotted. In FIG. 14B,C57BL6 mice bearing 5T33P myeloma tumors were treated with PBS,suboptimal ALT-803 (0.05 mg/kg), suboptimal anti-PD-L1 Ab (5 μg) orcombination ALT-803+anti-PD-L1 Ab as indicated in the figure. Thesurvival of the mice was monitored and Kaplan-Meier survival curves wereplotted.

FIG. 15A and FIG. 15B are a series of graphs showing the expression ofligands for PD1 and CTLA4 on the surface of tumor cells. CT26 (FIG. 15A)and 5T33P (FIG. 15B) tumor cells were stained with antibodies to PD-L1,CD86 and CD80 (red line) or isotype controls (black line) and thenanalyzed by follow cytometry.

FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, and FIG. 16F are aseries of bar graphs showing changes in mice observed followingmultidose treatment with ALT-803 and post-treatment recovery.

FIG. 17 is a line graph showing the pharmacokinetic profile of ALT-803in cynomolgus monkeys.

FIG. 18A-FIG. 18H are a series of line graphs showing changes in immunecell counts following multidose ALT-803 treatment and post-treatmentrecovery in cynomolgus monkeys.

FIG. 19A and FIG. 19B is a series of line graphs showing prolongedsurvival of mice bearing orthotopic MB49luc bladder tumors followingALT-803 plus checkpoint blockade therapy. In FIG. 19A, C57BL6 micebearing orthotopic MB49luc bladder tumors were treated with PBS,ALT-803, and ALT-803 combination therapies with anti-CTLA4 Ab andanti-PD-L1 Ab as indicated in the figure. The survival of the mice wasmonitored and Kaplan-Meier survival curves were plotted. After 80 days,surviving mice of the ALT-803+anti-PD-L1/anti-CTLA4-Ab group andtreatment naïve age-matched mice were re-challenged with intravesicularadministration of MB49luc tumor cells. Mouse survival was furthermonitored. In FIG. 19B, C57BL6 mice bearing MB49luc bladder tumors weretreated with PBS, ALT-803, anti-PD-1 Ab, anti-CTLA-4 Ab or combinationtherapy as indicated in the figure. The survival of the mice wasmonitored and Kaplan-Meier survival curves were plotted.

FIG. 20 is a series of flow cytometry graphs showing the expression ofligands for PD1 and CTLA4 on the surface of MB49luc tumor cells. MB49luctumor cells were stained with antibodies to PD-L1, CD86 and CD80 (blueline) or isotype controls (black line) and then analyzed by followcytometry.

FIG. 21A-FIG. 21C is a series of line graphs showing the combinatorialeffect of ALT-803 and TA99 in syngeneic murine melanoma model. In FIG.21A, B16F10 melanoma cells (2×10⁵) were injected subcutaneously into theflank of C57BL/6 mice. Once palpable tumors were formed, mice wererandomized and treated intravenously with 0.2 mg/kg ALT-803, 10 mg/kgTA99, a combination of ALT-803+TA99, or PBS control on study day 10, 14,17, 21 and 24. FIG. 21B and FIG. 21C show the evaluation of the effectorfunctions of different cell subsets involved in the anti-tumor immunityof combined therapy. Depletion of CD4⁺ and CD8⁺ T cells was eachaccomplished by intraperitoneal injection of rat mAb GK1.5 (anti-CD4)and 53.6.72 (anti-CD8a), respectively. NK cell depletion was achieved byintraperitoneal administration of murine mAb PK136 (anti-NK1.1). For thedepletion of macrophages, mice were intraperitoneally injected withclodronate-loaded liposomes (Clophosome). The impact of depletion wasassessed using both tumor growth (FIG. 21B) and mice survival (FIG.21C). The survival curves show the study days when animals died due totumor metastasis or the tumor reached the threshold size (onedimension >20 mm) for tumor burden. n=8/group. **: p<0.01; ***: p<0.001.

FIG. 22A-FIG. 22D is a series of bar charts demonstratingALT-803-mediated increase immune cells in the spleen and tumormicroenvironment. In FIG. 22A-FIG. 22D, mice (n=6) were injected(subcutaneously) with B16F10 melanoma cells (2×10⁵) on study day 0 (SD0)and treated intraveneously with TA99, ALT-803, a combination ofALT-803+TA99 or PBS on SD17. On SD20, the percentages of CD8⁺ T cells(CD8a⁺), CD4⁺ T cells (CD4⁺), NK cells (panNK⁺), B cells (CD19⁺), andmacrophages (F4/80⁺) were quantified in splenocytes (FIG. 22A) and TILs(7-AAD-CD45⁺, FIG. 22B) using flow cytometry. The percentages ofCD8⁺CD44^(high) memory T cells among the CD8⁺ T cell population of thespleen (FIG. 22C) and TIL (FIG. 22D) were measured and plotted.

FIG. 23A-FIG. 23C is a series of line graphs demonstrating thatALT-803+TA99 provides immune protection from tumor rechallenge. In FIG.23A-FIG. 23B, mice (n=20) implanted subcutaneously with B16F10 cells(2×10⁵) on study day 0 (SD0) were immediately treated intravenously withPBS, TA99, or TA99+ALT-803. After three weeks of treatment and twomonths of monitoring, naïve and survivor animals were rechallengedcontralaterally by subcutaneous injection of B16F10 cells (2×10⁵).Tumor-free survival (animals maintaining a subcutaneous tumor mass<50mm³) of animals during initial tumor challenge (FIG. 23A) and tumorgrowth during rechallenge (FIG. 23B) were measured and plotted.TB=tumor-bearing. *: p<0.05; **: p<0.01; ***: p<0.001. In FIG. 23C,murine melanoma B16F10 cells (2×10⁵/mouse) were injected subcutaneouslyinto the flank of C57BL6 mice on study day-58 (SD-58). The mice wereinjected with B16F10 cells as in FIG. 23A and treated with ALT-803 (0.2mg/kg) and TA99 (10 mg/kg) intravenously twice a week for three weeksstarting on day 0. To deplete CD4⁺ T cells, CD8⁺ T cells, or NK cells,anti-CD4 (GK1.5), anti-CD8 (53-6.72) and/or anti-NK (PK136) wereadministrated intraperitoneally into the tumor-free mice 46 days posttumor inoculation. In order to assess anti-tumor memory response of thetumor-free mice, B16F10 cells (2×10⁵/mouse) were subcutaneously injectedcontralaterally into the tumor free mice 58 days (SD0) post the firsttumor inoculation. Treatment naïve mice injected B16F10 cells withserved as a control. Survival curve summarizes the study days whenanimals died due to tumor metastasis or the threshold size (tumorvolume>4000 cubic mm). n=10/group.

FIG. 24A-FIG. 24E is a series of bar charts showing that ALT-803activates CD4⁺ T cells and upregulates their PD-L1 expression, butlowers PD-1 expression on CD8⁺ T cells. CD4⁺ T cells (CD4⁺) from the TIL(7-AAD-CD45⁺) fraction (FIG. 24A and FIG. 24C) and spleen (FIG. 24B) oftumor-bearing mice (n=6) were stained with anti-CD25 (FIG. 24A) oranti-PD-L1 (FIG. 24B and FIG. 24C) antibodies three days after a singleinjection of test articles, followed by flow cytometry quantification.CD8⁺ T cells (CD8⁺; FIG. 24D and FIG. 24E) from the spleen (FIG. 24D)and TIL (7-AAD-CD45⁺) fraction (FIG. 24E) of tumor-bearing mice (n=6)were stained with anti-PD-1 (FIG. 24D and FIG. 24E) antibody three daysafter a single injection of test articles, followed by flow cytometryquantification. Expression of PD-L1 and PD-1 is scored using meanfluorescence intensity (MFI). *: p<0.05; **: p<0.01; ***: p<0.001.

FIG. 25A-FIG. 25D is a series of bar charts showing that ALT-803activates NK cells and downregulates PD-1 expression on NK cells. NKcells (panNK⁺) from the spleen (FIG. 25A and FIG. 25C) and TIL(7-AAD-CD45⁺) fraction (FIG. 25B and FIG. 25D) of tumor-bearing mice(n=6) were stained with anti-KLRG1 (FIG. 25A and FIG. 25B) and anti-PD-1(FIG. 25C and FIG. 25D) antibodies three days after a single injectionof test articles, followed by flow cytometry quantification. Expressionof KLRG1 and PD-1 is scored using mean fluorescence intensity (MFI). *:p<0.05; **: p<0.01; ***: p<0.001.

FIG. 26A-FIG. 26B is a series of line graphs showing the combinatorialeffect of ALT-803/TA99 and anti-PD-L1 mAb in syngeneic murine melanomamodel. In FIG. 26A, B16F10 melanoma cells (2×10⁵) were injectedsubcutaneously into the right dorsal flank of C57BL6 mice. Once palpabletumors were formed, mice were randomized and treated with 0.2 mg/kgALT-803 (i.v.) and 10 mg/kg TA99 (i.v.), with or without 100 μg/mouseanti-PD-L1 Ab 10F.9G2 (i.p.) on study day 10, 14, 17, 21 and 24. *:p<0.05; ***: p<0.001. FIG. 26B shows in vitro and in vivo expression ofPD-L1 on B16F10 cells. B16F10 cells harvested from in vitro culture(solid lines) as well as tumor-bearing mice (dashed lines) were stainedwith fluorophore-labeled anti-PD-L1 antibody (red) and subjected to flowcytometry. Antibody isotype (black) was included as negative control.

DETAILED DESCRIPTION

The invention is based, at least in part, on the surprising discoverythat an antibody in combination with ALT-803, a complex of aninterleukin-15 (IL-15) superagonist mutant and a dimeric IL-15 receptorα/Fc fusion protein, is useful for enhancing an immune response againsta neoplasia (e.g., a glioblastoma, prostate cancer, hematologicalcancer, B-cell neoplasms, multiple myeloma, B-cell lymphoma, Hodgkin'slymphoma, acute myeloid leukemia, chronic lymphocytic leukemia,cutaneous T-cell lymphoma, T-cell lymphoma, a solid tumor,urothelial/bladder carcinoma, melanoma, lung cancer, renal cellcarcinoma, breast cancer, head and neck cancer, colorectal cancer, andovarian cancer) or an infection (e.g., an infection with humanimmunodeficiency virus).

ALT-803

ALT-803 comprises an IL-15 mutant with increased ability to bind IL-2Rβγand enhanced biological activity (U.S. Pat. No. 8,507,222, incorporatedherein by reference). This super agonist mutant of IL-15 was describedin a publication (J Immunol 2009 183:3598) and a patent has been issuedby the U.S. Patent & Trademark Office on the super agonist and severalpatents applications are pending (e.g., U.S. Ser. Nos. 12/151,980 and13/238,925). This IL-15 super agonist in combination with a solubleIL-15a receptor fusion protein (IL-15RαSu/Fc) results in a proteincomplex with highly potent IL-15 activity in vitro and in vivo (Han etal., 2011, Cytokine, 56: 804-810; Xu, et al., 2013 Cancer Res.73:3075-86, Wong, et al., 2013, OncoImmunology 2:e26442). This IL-15super agonist complex (IL-15N72D:IL-15RαSu/Fc) is referred to asALT-803. Pharmacokinetic analysis indicated that the complex has ahalf-life in mice of 25 hours following i.v. administration. ALT-803exhibits impressive anti-tumor activity against aggressive solid andhematological tumor models in immunocompetent mice. It can beadministered as a monotherapy using a twice weekly or weekly i.v. doseregimen or as combinatorial therapy with an antibody. The ALT-803anti-tumor response is also durable. Tumor-bearing mice that were curedafter ALT-803 treatment were also highly resistant to re-challenge withthe same tumor cells indicating that ALT-803 induces effectiveimmunological memory responses against the re-introduced tumor cells.

Interleukin-15

Interleukin-15 (IL-15) is an important cytokine for the development,proliferation, and activation of effector NK cells and CD8⁺ memory Tcells. IL-15 binds to the IL-15 receptor α (IL-15Rα) and is presented intrans to the IL-2/IL-15 receptor β-common γ chain (IL-15Rβγ c) complexon effector cells. IL-15 and IL-2 share binding to the IL-15Rβγ_(c), andsignal through STAT3 and STAT5 pathways. However, IL-2 also supportsmaintenance of CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg) cells and inducescell death of activated CD8⁺ T cells. These effects may limit thetherapeutic activity of IL-2 against tumors. IL-15 does not share theseimmunosurppresive activities with IL-2. Additionally, IL-15 is the onlycytokine known to provide anti-apoptotic signaling to effector CD8⁺ Tcells. IL-15, either administered alone or as a complex with theIL-15Rα, exhibits potent anti-tumor activities against well-establishedsolid tumors in experimental animal models and, thus, has beenidentified as one of the most promising immunotherapeutic drugs thatcould potentially cure cancer.

To facilitate clinical development of an IL-15-based cancer therapeutic,an IL-15 mutant (IL-15N72D) with increased biological activity comparedto IL-15 was identified (Zhu et al., J Immunol, 183: 3598-3607, 2009).The pharmacokinetics and biological activity of this IL-15 super-agonist(IL-15N72D) was further improved by the creation ofIL-15N72D:IL-15RαSu/Fc fusion complex (ALT-803), such that the superagonist complex has at least 25-times the activity of the nativecytokine in vivo (Han et al., Cytokine, 56: 804-810, 2011).

Fc Domain

ALT-803 comprises an IL-15N72D:IL-15RαSu/Fc fusion complex. Fusionproteins that combine the Fc regions of IgG with the domains of anotherprotein, such as various cytokines and soluble receptors have beenreported (see, for example, Capon et al., Nature, 337:525-531, 1989;Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Pat. Nos.5,116,964 and 5,541,087). The prototype fusion protein is a homodimericprotein linked through cysteine residues in the hinge region of IgG Fc,resulting in a molecule similar to an IgG molecule without the heavychain variable and C_(H1) domains and light chains. The dimeric natureof fusion proteins comprising the Fc domain may be advantageous inproviding higher order interactions (i.e. bivalent or bispecificbinding) with other molecules. Due to the structural homology, Fc fusionproteins exhibit an in vivo pharmacokinetic profile comparable to thatof human IgG with a similar isotype. Immunoglobulins of the IgG classare among the most abundant proteins in human blood, and theircirculation half-lives can reach as long as 21 days. To extend thecirculating half-life of IL-15 or an IL-15 fusion protein and/or toincrease its biological activity, fusion protein complexes containingthe IL-15 domain non-covalently bound to IL-15RαSu covalently linked tothe Fc portion of the human heavy chain IgG protein have been made(e.g., ALT-803).

The term “Fc” refers to a non-antigen-binding fragment of an antibody.Such an “Fc” can be in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulins, although IgG 1 and IgG2 arepreferred. Native Fc's are made up of monomeric polypeptides that may belinked into dimeric or multimeric forms by covalent (i.e., disulfidebonds) and non-covalent association. The number of intermoleculardisulfide bonds between monomeric subunits of native Fc molecules rangesfrom 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is adisulfide-bonded dimer resulting from papain digestion of an IgG (seeEllison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “nativeFc” as used herein is generic to the monomeric, dimeric, and multimericforms. Fc domains containing binding sites for Protein A, Protein G,various Fc receptors and complement proteins.

In some embodiments, the term “Fc variant” refers to a molecule orsequence that is modified from a native Fc, but still comprises abinding site for the salvage receptor, FcRn. International applicationsWO 97/34631 (published Sep. 25, 1997) and WO 96/32478 describe exemplaryFc variants, as well as interaction with the salvage receptor, and arehereby incorporated by reference. Thus, the term “Fc variant” comprisesa molecule or sequence that is humanized from a non-human native Fc.Furthermore, a native Fc comprises sites that may be removed becausethey provide structural features or biological activity that are notrequired for the fusion molecules of the present invention. Thus, incertain embodiments, the term “Fc variant” comprises a molecule orsequence that lacks one or more native Fc sites or residues that affector are involved in (1) disulfide bond formation, (2) incompatibilitywith a selected host cell (3)N-terminal heterogeneity upon expression ina selected host cell, (4) glycosylation, (5) interaction withcomplement, (6) binding to an Fc receptor other than a salvage receptor,(7) antibody-dependent cell-mediated cytotoxicity (ADCC), or (8)antibody dependent cellular phagocytosis (ADCP). Fc variants aredescribed in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by recombinant geneexpression or by other means.

Fusions Protein Complexes

The invention provides ALT-803, which is a protein complex betweenIL-15N72D and IL-15RαSu/Fc. In certain embodiments, the ALT-803polypeptides could serve as a scaffold for fusion to other proteindomains. In such fusion protein complexes, a first fusion proteincomprises a first biologically active polypeptide covalently linked tointerleukin-15 (IL-15) or functional fragment thereof, and the secondfusion protein comprises a second biologically active polypeptidecovalently linked to soluble interleukin-15 receptor alpha (IL-15Rα)polypeptide or functional fragment thereof, where the IL-15 domain of afirst fusion protein binds to the soluble IL-15Rα domain of the secondfusion protein to form a soluble fusion protein complex. Fusion proteincomplexes of the invention also comprise immunoglobulin Fc domain or afunctional fragment thereof linked to one or both of the first andsecond fusion proteins. Preferably, the Fc domains linked to the firstand second fusion proteins interact to form a fusion protein complex.Such a complex may be stabilized by disulfide bond formation between theimmunoglobulin Fc domains. In certain embodiments, the soluble fusionprotein complexes of the invention include an IL-15 polypeptide, IL-15variant or a functional fragment thereof and a soluble IL-15Rαpolypeptide or a functional fragment thereof, wherein one or both of theIL-15 and IL-15Rα polypeptides further include an immunoglobulin Fcdomain or a functional fragment thereof.

In a further embodiment, one or both of the first and secondbiologically active polypeptides comprises an antibody or functionalfragment thereof.

In another embodiment, the antigen for the antibody domain comprises acell surface receptor or ligand.

In a further embodiment, the antigen comprises a CD antigen, cytokine orchemokine receptor or ligand, growth factor receptor or ligand, tissuefactor, cell adhesion molecule, MHC/MHC-like molecules, Fc receptor,Toll-like receptor, NK receptor, TCR, BCR, positive/negativeco-stimulatory receptor or ligand, death receptor or ligand, tumorassociated antigen, or virus encoded antigen.

As used herein, the term “biologically active polypeptide” or “effectormolecule” is meant an amino acid sequence such as a protein, polypeptideor peptide; a sugar or polysaccharide; a lipid or a glycolipid,glycoprotein, or lipoprotein that can produce the desired effects asdiscussed herein. Effector molecules also include chemical agents. Alsocontemplated are effector molecule nucleic acids encoding a biologicallyactive or effector protein, polypeptide, or peptide. Thus, suitablemolecules include regulatory factors, enzymes, antibodies, or drugs aswell as DNA, RNA, and oligonucleotides. The biologically activepolypeptides or effector molecule can be naturally-occurring or it canbe synthesized from known components, e.g., by recombinant or chemicalsynthesis and can include heterologous components. A biologically activepolypeptides or effector molecule is generally between about 0.1 to 100KD or greater up to about 1000 KD, preferably between about 0.1, 0.2,0.5, 1, 2, 5, 10, 20, 30 and 50 KD as judged by standard molecule sizingtechniques such as centrifugation or SDS-polyacrylamide gelelectrophoresis. Desired effects of the invention include, but are notlimited to, for example, forming a fusion protein complex of theinvention with increased binding activity, killing a target cell, e.g.either to induce cell proliferation or cell death, initiate an immuneresponse, in preventing or treating a disease, or to act as a detectionmolecule for diagnostic purposes. For such detection, an assay could beused, for example an assay that includes sequential steps of culturingcells to proliferate same.

Covalently linking the effector molecule to the fusion protein complexesof the invention in accordance with the invention provides a number ofsignificant advantages. Fusion protein complexes of the invention can beproduced that contain a single effector molecule, including such apeptide of known structure. Additionally, a wide variety of effectormolecules can be produced in similar DNA vectors. That is, a library ofdifferent effector molecules can be linked to the fusion proteincomplexes for recognition of infected or diseased cells. Further, fortherapeutic applications, rather than administration of a the fusionprotein complex of the invention to a subject, a DNA expression vectorcoding for the fusion protein complex can be administered for in vivoexpression of the fusion protein complex. Such an approach avoids costlypurification steps typically associated with preparation of recombinantproteins and avoids the complexities of antigen uptake and processingassociated with conventional approaches.

As noted, components of the fusion proteins and antibodies disclosedherein, e.g., effector molecule conjugates such as cytokines,chemokines, growth factors, protein toxins, immunoglobulin domains orother bioactive molecules and any peptide linkers, can be organized innearly any fashion provided that the fusion protein or antibody has thefunction for which it was intended. In particular, each component of thefusion protein can be spaced from another component by at least onesuitable peptide linker sequence if desired. Additionally, the fusionproteins may include tags, e.g., to facilitate modification,identification and/or purification of the fusion protein.

Pharmaceutical Therapeutics

The invention provides pharmaceutical compositions comprising ALT-803for use as a therapeutic. In one aspect, ALT-803 is administeredsystemically, for example, formulated in a pharmaceutically-acceptablebuffer such as physiological saline. Preferable routes of administrationinclude, for example, instillation into the bladder, subcutaneous,intravenous, intraperitoneal, intramuscular, or intradermal injectionsthat provide continuous, sustained levels of the composition in thepatient. Treatment of human patients or other animals is carried outusing a therapeutically effective amount of a therapeutic identifiedherein in a physiologically-acceptable carrier. Suitable carriers andtheir formulation are described, for example, in Remington'sPharmaceutical Sciences by E. W. Martin. The amount of the therapeuticagent to be administered varies depending upon the manner ofadministration, the age and body weight of the patient, and with theclinical symptoms of the neoplasia or infection. Generally, amounts willbe in the range of those used for other agents used in the treatment ofother diseases associated with neoplasia or infection, although incertain instances lower amounts will be needed because of the increasedspecificity of the compound. A compound is administered at a dosage thatenhances an immune response of a subject, or that reduces theproliferation, survival, or invasiveness of a neoplastic cell asdetermined by a method known to one skilled in the art. Alternatively,the compound is administered at a dosage that reduces infection by avirus or other pathogen of interest.

Formulation of Pharmaceutical Compositions

The administration of ALT-803 for the treatment of a neoplasia or aninfection may be by any suitable means that results in a concentrationof the therapeutic that, combined with other components, is effective inameliorating, reducing, or stabilizing a neoplasia or infection. ALT-803may be contained in any appropriate amount in any suitable carriersubstance, and is generally present in an amount of 1-95% by weight ofthe total weight of the composition. The composition may be provided ina dosage form that is suitable for parenteral (e.g., subcutaneously,intravenously, intramuscularly, intravesicularly or intraperitoneally)administration route. The pharmaceutical compositions may be formulatedaccording to conventional pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2000 and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York).

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice or nonhuman primates, as a skilledartisan recognizes it is routine in the art to modify the dosage forhumans compared to animal models. In certain embodiments it isenvisioned that the dosage may vary from between about 0.1 μgcompound/kg body weight to about 5000 μg compound/kg body weight; orfrom about 1 μg/kg body weight to about 4000 μg/kg body weight or fromabout 10 μg/kg body weight to about 3000 μg/kg body weight. In otherembodiments this dose may be about 0.1, 0.3, 0.5, 1, 3, 5, 10, 25, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, or 5000 μg/kg body weight. In other embodiments, it is envisagedthat doses may be in the range of about 0.5 μg compound/kg body weightto about 20 μg compound/kg body weight. In other embodiments the dosesmay be about 0.5, 1, 3, 6, 10, or 20 mg/kg body weight. Of course, thisdosage amount may be adjusted upward or downward, as is routinely donein such treatment protocols, depending on the results of the initialclinical trials and the needs of a particular patient.

In particular embodiments, ALT-803 are formulated in an excipientsuitable for parenteral administration. In particular embodiments,ALT-803 is administered at 0.5 μg/kg-about 15 μg/kg (e.g., 0.5, 1, 3, 5,10, or 15 μg/kg).

For the treatment of bladder cancer, ALT-803 is administered byinstillation into the bladder. Methods of instillation are known. See,for example, Lawrencia, et al., Gene Ther 8, 760-8 (2001); Nogawa, etal., J Clin Invest 115, 978-85 (2005); Ng, et al., Methods Enzymol 391,304-13 2005; Tyagi, et al., J Urol 171, 483-9 (2004); Trevisani, et al.,J Pharmacol Exp Ther 309, 1167-73 (2004); Trevisani, et al., NatNeurosci 5, 546-51 (2002)); (Segal, et al., 1975). (Dyson, et al.,2005). (Batista, et al., 2005; Dyson, et al., 2005). In certainembodiments, it is envisioned that the ALT-803 dosage for instillationmay vary from between about 5 and 1000 μg/dose. In other embodiments theintravesical doses may be about 25, 50, 100, 200, or 400 μg/dose. Inother embodiments, ALT-803 is administered by instillation into thebladder in combination with standard thereapies, including mitomycin Cor Bacille Calmette-Guerin (BCG).

Pharmaceutical compositions are formulated with appropriate excipientsinto a pharmaceutical composition that, upon administration, releasesthe therapeutic in a controlled manner. Examples include single ormultiple unit tablet or capsule compositions, oil solutions,suspensions, emulsions, microcapsules, microspheres, molecularcomplexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition comprising ALT-803 may be administeredparenterally by injection, infusion or implantation (subcutaneous,intravenous, intramuscular, intravesicularly, intraperitoneal, or thelike) in dosage forms, formulations, or via suitable delivery devices orimplants containing conventional, non-toxic pharmaceutically acceptablecarriers and adjuvants. The formulation and preparation of suchcompositions are well known to those skilled in the art ofpharmaceutical formulation. Formulations can be found in Remington: TheScience and Practice of Pharmacy, supra.

Compositions comprising ALT-803 for parenteral use may be provided inunit dosage forms (e.g., in single-dose ampoules, syringes or bags), orin vials containing several doses and in which a suitable preservativemay be added (see below). The composition may be in the form of asolution, a suspension, an emulsion, an infusion device, or a deliverydevice for implantation, or it may be presented as a dry powder to bereconstituted with water or another suitable vehicle before use. Apartfrom the active agent that reduces or ameliorates a neoplasia orinfection, the composition may include suitable parenterally acceptablecarriers and/or excipients. The active therapeutic agent(s) may beincorporated into microspheres, microcapsules, nanoparticles, liposomes,or the like for controlled release. Furthermore, the composition mayinclude suspending, solubilizing, stabilizing, pH-adjusting agents,tonicity adjusting agents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions comprising ALT-803may be in a form suitable for sterile injection. To prepare such acomposition, the suitable active antineoplastic/anti-infectivetherapeutic(s) are dissolved or suspended in a parenterally acceptableliquid vehicle. Among acceptable vehicles and solvents that may beemployed are water, water adjusted to a suitable pH by addition of anappropriate amount of hydrochloric acid, sodium hydroxide or a suitablebuffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloridesolution and dextrose solution. The aqueous formulation may also containone or more preservatives (e.g., methyl, ethyl or n-propylp-hydroxybenzoate). In cases where one of the compounds is onlysparingly or slightly soluble in water, a dissolution enhancing orsolubilizing agent can be added, or the solvent may include 10-60% w/wof propylene glycol or the like.

The present invention provides methods of treating neoplastic orinfectious disease and/or disorders or symptoms thereof which compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound of the formulae herein to a subject(e.g., a mammal such as a human). Thus, one embodiment is a method oftreating a subject suffering from or susceptible to a neoplastic orinfectious disease or disorder or symptom thereof. The method includesthe step of administering to the mammal a therapeutic amount of anamount of a compound herein sufficient to treat the disease or disorderor symptom thereof, under conditions such that the disease or disorderis treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a neoplastic or infectiousdisease, disorder, or symptom thereof. Determination of those subjects“at risk” can be made by any objective or subjective determination by adiagnostic test or opinion of a subject or health care provider (e.g.,genetic test, enzyme or protein marker, Marker (as defined herein),family history, and the like). ALT-803 may be used in the treatment ofany other disorders in which an increase in an immune response isdesired.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof associated withneoplasia or infection in which the subject has been administered atherapeutic amount of a compound herein sufficient to treat the diseaseor symptoms thereof. The level of Marker determined in the method can becompared to known levels of Marker in either healthy normal controls orin other afflicted patients to establish the subject's disease status.In preferred embodiments, a second level of Marker in the subject isdetermined at a time point later than the determination of the firstlevel, and the two levels are compared to monitor the course of diseaseor the efficacy of the therapy. In certain preferred embodiments, apre-treatment level of Marker in the subject is determined prior tobeginning treatment according to this invention; this pre-treatmentlevel of Marker can then be compared to the level of Marker in thesubject after the treatment commences, to determine the efficacy of thetreatment.

Combination Therapies

Preferably, ALT-803 is administered in combination with ananti-neoplasia or anti-infectious therapeutic such as an antibody, e.g.,a tumor-specific antibody or an immune-checkpoint inhibitor. Theantibody and ALT-803 may be administered simultaneously or sequentially.In some embodiments, the antibody treatment is an established therapyfor the disease indication and addition of ALT-803 treatment to theantibody regimen improves the therapeutic benefit to the patients. Suchimprovement could be measured as increased responses on a per patientbasis or increased responses in the patient population. Combinationtherapy could also provide improved responses at lower or less frequentdoses of antibody resulting in a better tolerated treatment regimen. Asindicated, the combined therapy of ALT-803 and an antibody could provideenhances clinical activity through various mechanisms, includingaugmented ADCC, ADCP, and/or NK cell, T-cell, neutrophil or monocyticcell levels or immune responses.

If desired, ALT-803 is administered in combination with any conventionaltherapy, including but not limited to, surgery, radiation therapy,chemotherapy, protein-based therapy or biological therapy.Chemotherapeutic drugs include alkylating agents (e.g., platinum-baseddrugs, tetrazines, aziridines, nitrosoureas, nitrogen mustards),anti-metabolites (e.g., anti-folates, fluoropyrimidines, deoxynucleosideanalogues, thiopurines), anti-microtubule agents (e.g., vinca alkaloids,taxanes), topoisomerase inhibitors (e.g., topoisomerase I and IIinhibitors), cytotoxic antibiotics (e.g., anthracyclines) andimmunomodulatory drugs (e.g., thalidomide and analogs).

Kits or Pharmaceutical Systems

Pharmaceutical compositions comprising ALT-803 may be assembled intokits or pharmaceutical systems for use in treating a neoplasia orinfection. Kits or pharmaceutical systems according to this aspect ofthe invention comprise a carrier means, such as a box, carton, tube,having in close confinement therein one or more container means, such asvials, tubes, ampoules, bottles, syringes, or bags. The kits orpharmaceutical systems of the invention may also comprise associatedinstructions for using ALT-803.

Recombinant Protein Expression

In general, preparation of the fusion protein complexes of the invention(e.g., components of ALT-803) can be accomplished by proceduresdisclosed herein and by recognized recombinant DNA techniques.

In general, recombinant polypeptides are produced by transformation of asuitable host cell with all or part of a polypeptide-encoding nucleicacid molecule or fragment thereof in a suitable expression vehicle.Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. A recombinant polypeptide may be produced in virtually anyeukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g.,Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS or preferablyCHO cells). Such cells are available from a wide range of sources (e.g.,the American Type Culture Collection, Rockland, Md.; also, see, e.g.,Ausubel et al., Current Protocol in Molecular Biology, New York: JohnWiley and Sons, 1997). The method of transfection and the choice ofexpression vehicle will depend on the host system selected.Transformation methods are described, e.g., in Ausubel et al. (supra);expression vehicles may be chosen from those provided, e.g., in CloningVectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of recombinantpolypeptides. Expression vectors useful for producing such polypeptidesinclude, without limitation, chromosomal, episomal, and virus-derivedvectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertionelements, from yeast chromosomal elements, from viruses such asbaculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof.

Once the recombinant polypeptide is expressed, it is isolated, e.g.,using affinity chromatography. In one example, an antibody (e.g.,produced as described herein) raised against the polypeptide may beattached to a column and used to isolate the recombinant polypeptide.Lysis and fractionation of polypeptide-harboring cells prior to affinitychromatography may be performed by standard methods (see, e.g., Ausubelet al., supra). Once isolated, the recombinant protein can, if desired,be further purified, e.g., by high performance liquid chromatography(see, e.g., Fisher, Laboratory Techniques In Biochemistry and MolecularBiology, eds., Work and Burdon, Elsevier, 1980).

As used herein, biologically active polypeptides or effector moleculesof the invention may include factors such as cytokines, chemokines,growth factors, protein toxins, immunoglobulin domains or otherbioactive proteins such as enzymes. Also biologically activepolypeptides may include conjugates to other compounds such asnon-protein toxins, cytotoxic agents, chemotherapeutic agents,detectable labels, radioactive materials and such.

Cytokines of the invention are defined by any factor produced by cellsthat affect other cells and are responsible for any of a number ofmultiple effects of cellular immunity. Examples of cytokines include butare not limited to the IL-2 family, interferon (IFN), IL-10, IL-1,IL-17, TGF and TNF cytokine families, and to IL-1 through IL-35, IFN-α,IFN-β, IFNγ, TGF-β, TNF-α, and TNFβ.

In an aspect of the invention, the first fusion protein comprises afirst biologically active polypeptide covalently linked tointerleukin-15 (IL-15) domain or a functional fragment thereof. IL-15 isa cytokine that affects T-cell activation and proliferation. IL-15activity in affecting immune cell activation and proliferation issimilar in some respects to IL-2, although fundamental differences havebeen well characterized (Waldmann, T A, 2006, Nature Rev. Immunol.6:595-601).

In another aspect of the invention, the first fusion protein comprisesan interleukin-15 (IL-15) domain that is an IL-15 variant (also referredto herein as IL-15 mutant). The IL-15 variant preferably comprises adifferent amino acid sequence that the native (or wild type) IL-15protein. The IL-15 variant preferably binds the IL-15Rα polypeptide andfunctions as an IL-15 agonist or antagonist. Preferably, IL-15 variantswith agonist activity have super agonist activity. In some embodiments,the IL-15 variant can function as an IL-15 agonist or antagonistindependent of its association with IL-15Rα. IL-15 agonists areexemplified by comparable or increased biological activity compared towild type IL-15. IL-15 antagonists are exemplified by decreasedbiological activity compared to wild type IL-15 or by the ability toinhibit IL-15-mediated responses. In some examples, the IL-15 variantbinds with increased or decreased activity to the IL-15RβγC receptors.In some embodiments, the sequence of the IL-15 variant has at least oneamino acid change, e.g. substitution or deletion, compared to the nativeIL-15 sequence, such changes resulting in IL-15 agonist or antagonistactivity. Preferably the amino acid substitutions/deletions are in thedomains of IL-15 that interact with IL-15Rβ and/or γC. More preferably,the amino acid substitutions/deletions do not affect binding to theIL-15Rα polypeptide or the ability to produce the IL-15 variant.Suitable amino acid substitutions/deletions to generate IL-15 variantscan be identified based on putative or known IL-15 structures,comparisons of IL-15 with homologous molecules such as IL-2 with knownstructure, through rational or random mutagenesis and functional assays,as provided herein, or other empirical methods. Additionally suitableamino acid substitutions can be conservative or non-conservative changesand insertions of additional amino acids. Preferably, IL-15 variants ofthe invention contain one or more than one amino acidsubstitutions/deletions at position 6, 8, 10, 61, 65, 72, 92, 101, 104,105, 108, 109, 111, or 112 of the mature human IL-15 sequence;particularly, D8N (“D8” refers to the amino acid and residue position inthe native mature human IL-15 sequence and “N” refers to the substitutedamino acid residue at that position in the IL-15 variant), I6S, D8A,D61A, N65A, N72R, V104P or Q108A substitutions result in IL-15 variantswith antagonist activity and N72D substitutions result in IL-15 variantswith agonist activity.

Chemokines, similar to cytokines, are defined as any chemical factor ormolecule which when exposed to other cells are responsible for any of anumber of multiple effects of cellular immunity. Suitable chemokines mayinclude but are not limited to the CXC, CC, C, and CX.sub.3C chemokinefamilies and to CCL-1 through CCL-28, CXC-1 through CXC-17, XCL-1,XCL-2, CX3CL1, MIP-1b, IL-8, MCP-1, and Rantes.

Growth factors include any molecules which when exposed to a particularcell induce proliferation and/or differentiation of the affected cell.Growth factors include proteins and chemical molecules, some of whichinclude: GM-CSF, G-CSF, human growth factor and stem cell growth factor.Additional growth factors may also be suitable for uses describedherein.

Toxins or cytotoxic agents include any substance that has a lethaleffect or an inhibitory effect on growth when exposed to cells. Morespecifically, the effector molecule can be a cell toxin of, e.g., plantor bacterial origin such as, e.g., diphtheria toxin (DT), shiga toxin,abrin, cholera toxin, ricin, saporin, pseudomonas exotoxin (PE),pokeweed antiviral protein, or gelonin. Biologically active fragments ofsuch toxins are well known in the art and include, e.g., DT A chain andricin A chain. Additionally, the toxin can be an agent active at thecell surface such as, e.g., phospholipase enzymes (e.g., phospholipaseC).

Further, the effector molecule can be a chemotherapeutic drug such as,e.g., vindesine, vincristine, vinblastin, methotrexate, adriamycin,bleomycin, or cisplatin.

Additionally, the effector molecule can be a detectably-labeled moleculesuitable for diagnostic or imaging studies. Such labels include biotinor streptavidin/avidin, a detectable nanoparticles or crystal, an enzymeor catalytically active fragment thereof, a fluorescent label such asgreen fluorescent protein, FITC, phycoerythrin, cychome, texas red orquantum dots; a radionuclide e.g., iodine-131, yttrium-90, rhenium-188or bismuth-212; a phosphorescent or chemiluminescent molecules or alabel detectable by PET, ultrasound or MRI such as Gd- or paramagneticmetal ion-based contrast agents. See e.g., Moskaug, et al. J. Biol.Chem. 264, 15709 (1989); Pastan, I. et al. Cell 47, 641, 1986; Pastan etal., Recombinant Toxins as Novel Therapeutic Agents, Ann. Rev. Biochem.61, 331, (1992); “Chimeric Toxins” Olsnes and Phil, Pharmac. Ther., 25,355 (1982); published PCT application no. WO 94/29350; published PCTapplication no. WO 94/04689; published PCT application no. WO2005046449and U.S. Pat. No. 5,620,939 for disclosure relating to making and usingproteins comprising effectors or tags.

A protein fusion or conjugate complex that includes a covalently linkedIL-15 and IL-15Rα domains has several important uses. Cells or tissuesusceptible to being damaged or killed can be readily assayed by themethods disclosed herein.

The IL-15 and IL-15Rα polypeptides of the invention suitably correspondin amino acid sequence to naturally occurring IL-15 and IL-15Rαmolecules, e.g. IL-15 and IL-15Rα molecules of a human, mouse or otherrodent, or other mammal. Sequences of these polypeptides and encodingnucleic acids are known in the literature, including human interleukin15 (IL15) mRNA—GenBank: U14407.1, Mus musculus interleukin 15 (IL15)mRNA—GenBank: U14332.1, human interleukin-15 receptor alpha chainprecursor (IL15RA) mRNA—GenBank: U31628.1, Mus musculus interleukin 15receptor, alpha chain—GenBank: BC095982.1.

In some settings, it can be useful to make the protein fusion orconjugate complexes of the present invention polyvalent, e.g., toincrease the valency of the sc-TCR or sc-antibody. In particular,interactions between the IL-15 and IL-15Rα domains of the fusion proteincomplex provide a means of generating polyvalent complexes. In addition,the polyvalent fusion protein can made by covalently or non-covalentlylinking together between one and four proteins (the same or different)by using e.g., standard biotin-streptavidin labeling techniques, or byconjugation to suitable solid supports such as latex beads. Chemicallycross-linked proteins (for example cross-linked to nanoparticles) arealso suitable polyvalent species. For example, the protein can bemodified by including sequences encoding tag sequences that can bemodified such as the biotinylation BirA tag or amino acid residues withchemically reactive side chains such as Cys or His. Such amino acid tagsor chemically reactive amino acids may be positioned in a variety ofpositions in the fusion protein or antibody, preferably distal to theactive site of the biologically active polypeptide or effector molecule.For example, the C-terminus of a soluble fusion protein can becovalently linked to a tag or other fused protein which includes such areactive amino acid(s). Suitable side chains can be included tochemically link two or more fusion proteins to a suitable nanoparticleto give a multivalent molecule. Exemplary nanoparticles includedendrimers, liposomes, core-shell particles or PLGA-based particles.

In another embodiment of the invention, one or both of the polypeptidesof the fusion protein complex comprises an immunoglobulin domain.Alternatively, the protein binding domain-IL-15 fusion protein can befurther linked to an immunoglobulin domain. The preferred immunoglobulindomains comprise regions that allow interaction with otherimmunoglobulin domains to form multichain proteins as provided above.For example, the immunoglobulin heavy chain regions, such as the IgG1C_(H2)-C_(H3), are capable of stably interacting to create the Fcregion. Preferred immunoglobulin domains including Fc domains alsocomprise regions with effector functions, including Fc receptor orcomplement protein binding activity, and/or with glycosylation sites. Insome embodiments, the immunoglobulin domains of the fusion proteincomplex contain mutations that reduce or augment Fc receptor orcomplement binding activity or glycosylation, thereby affecting thebiological activity of the resulting protein. For example,immunoglobulin domains containing mutations that reduce binding to Fcreceptors could be used to generate fusion protein complex of theinvention with lower binding activity to Fc receptor-bearing cells,which may be advantageous for reagents designed to recognize or detectspecific antigens.

Nucleic Acids and Vectors

The invention further provides nucleic acid sequences and particularlyDNA sequences that encode the present proteins (e.g., components ofALT-803). Preferably, the DNA sequence is carried by a vector suited forextrachromosomal replication such as a phage, virus, plasmid, phagemid,cosmid, YAC, or episome. In particular, a DNA vector that encodes adesired fusion protein can be used to facilitate preparative methodsdescribed herein and to obtain significant quantities of the fusionprotein. The DNA sequence can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein-coding sequence. Avariety of host-vector systems may be utilized to express theprotein-coding sequence. These include mammalian cell systems infectedwith virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophageDNA, plasmid DNA or cosmid DNA. Depending on the host-vector systemutilized, any one of a number of suitable transcription and translationelements may be used. See, Sambrook et al., supra and Ausubel et al.supra.

Included in the invention are methods for making a soluble fusionprotein complex, the method comprising introducing into a host cell aDNA vector as described herein encoding the first and second fusionproteins, culturing the host cell in media under conditions sufficientto express the fusion proteins in the cell or the media and allowassociation between IL-15 domain of a first fusion protein and thesoluble IL-15Rα domain of a second fusion protein to form the solublefusion protein complex, purifying the soluble fusion protein complexfrom the host cells or media.

In general, a preferred DNA vector according to the invention comprisesa nucleotide sequence linked by phosphodiester bonds comprising, in a 5′to 3′ direction a first cloning site for introduction of a firstnucleotide sequence encoding a biologically active polypeptide,operatively linked to a sequence encoding an effector molecule.

The fusion protein components encoded by the DNA vector can be providedin a cassette format. By the term “cassette” is meant that eachcomponent can be readily substituted for another component by standardrecombinant methods. In particular, a DNA vector configured in acassette format is particularly desirable when the encoded fusioncomplex is to be used against pathogens that may have or have capacityto develop serotypes.

To make the vector coding for a fusion protein complex, the sequencecoding for the biologically active polypeptide is linked to a sequencecoding for the effector peptide by use of suitable ligases. DNA codingfor the presenting peptide can be obtained by isolating DNA from naturalsources such as from a suitable cell line or by known synthetic methods,e.g. the phosphate triester method. See, e.g., OligonucleotideSynthesis, IRL Press (M. J. Gait, ed., 1984). Synthetic oligonucleotidesalso may be prepared using commercially available automatedoligonucleotide synthesizers. Once isolated, the gene coding for thebiologically active polypeptide can be amplified by the polymerase chainreaction (PCR) or other means known in the art. Suitable PCR primers toamplify the biologically active polypeptide gene may add restrictionsites to the PCR product. The PCR product preferably includes splicesites for the effector peptide and leader sequences necessary for properexpression and secretion of the biologically active polypeptide-effectorfusion complex. The PCR product also preferably includes a sequencecoding for the linker sequence, or a restriction enzyme site forligation of such a sequence.

The fusion proteins described herein are preferably produced by standardrecombinant DNA techniques. For example, once a DNA molecule encodingthe biologically active polypeptide is isolated, sequence can be ligatedto another DNA molecule encoding the effector polypeptide. Thenucleotide sequence coding for a biologically active polypeptide may bedirectly joined to a DNA sequence coding for the effector peptide or,more typically, a DNA sequence coding for the linker sequence asdiscussed herein may be interposed between the sequence coding for thebiologically active polypeptide and the sequence coding for the effectorpeptide and joined using suitable ligases. The resultant hybrid DNAmolecule can be expressed in a suitable host cell to produce the fusionprotein complex. The DNA molecules are ligated to each other in a 5′ to3′ orientation such that, after ligation, the translational frame of theencoded polypeptides is not altered (i.e., the DNA molecules are ligatedto each other in-frame). The resulting DNA molecules encode an in-framefusion protein.

Other nucleotide sequences also can be included in the gene construct.For example, a promoter sequence, which controls expression of thesequence coding for the biologically active polypeptide fused to theeffector peptide, or a leader sequence, which directs the fusion proteinto the cell surface or the culture medium, can be included in theconstruct or present in the expression vector into which the constructis inserted. An immunoglobulin or CMV promoter is particularlypreferred.

In obtaining variant biologically active polypeptide, IL-15, IL-15Rα orFc domain coding sequences, those of ordinary skill in the art willrecognize that the polypeptides may be modified by certain amino acidsubstitutions, additions, deletions, and post-translationalmodifications, without loss or reduction of biological activity. Inparticular, it is well-known that conservative amino acid substitutions,that is, substitution of one amino acid for another amino acid ofsimilar size, charge, polarity and conformation, are unlikely tosignificantly alter protein function. The 20 standard amino acids thatare the constituents of proteins can be broadly categorized into fourgroups of conservative amino acids as follows: the nonpolar(hydrophobic) group includes alanine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan and valine; the polar (uncharged,neutral) group includes asparagine, cysteine, glutamine, glycine,serine, threonine and tyrosine; the positively charged (basic) groupcontains arginine, histidine and lysine; and the negatively charged(acidic) group contains aspartic acid and glutamic acid. Substitution ina protein of one amino acid for another within the same group isunlikely to have an adverse effect on the biological activity of theprotein. In other instance, modifications to amino acid positions can bemade to reduce or enhance the biological activity of the protein. Suchchanges can be introduced randomly or via site-specific mutations basedon known or presumed structural or functional properties of targetedresidue(s). Following expression of the variant protein, the changes inthe biological activity due to the modification can be readily assessedusing binding or functional assays.

Homology between nucleotide sequences can be determined by DNAhybridization analysis, wherein the stability of the double-stranded DNAhybrid is dependent on the extent of base pairing that occurs.Conditions of high temperature and/or low salt content reduce thestability of the hybrid, and can be varied to prevent annealing ofsequences having less than a selected degree of homology. For instance,for sequences with about 55% G-C content, hybridization and washconditions of 40-50° C., 6×SSC (sodium chloride/sodium citrate buffer)and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology,hybridization and wash conditions of 50-65° C., 1×SSC and 0.1% SDSindicate about 82-97% homology, and hybridization and wash conditions of52° C., 0.1×SSC and 0.1% SDS indicate about 99-100% homology. A widerange of computer programs for comparing nucleotide and amino acidsequences (and measuring the degree of homology) are also available, anda list providing sources of both commercially available and freesoftware is found in Ausubel et al. (1999). Readily available sequencecomparison and multiple sequence alignment algorithms are, respectively,the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997)and ClustalW programs. BLAST is available on the world wide web atncbi.nlm.nih.gov and a version of ClustalW is available at 2.ebi.ac.uk.

The components of the fusion protein can be organized in nearly anyorder provided each is capable of performing its intended function. Forexample, in one embodiment, the biologically active polypeptide issituated at the C or N terminal end of the effector molecule.

Preferred effector molecules of the invention will have sizes conduciveto the function for which those domains are intended. The effectormolecules of the invention can be made and fused to the biologicallyactive polypeptide by a variety of methods including well-known chemicalcross-linking methods. See, e.g., Means, G. E. and Feeney, R. E. (1974)in Chemical Modification of Proteins, Holden-Day. See also, S. S. Wong(1991) in Chemistry of Protein Conjugation and Cross-Linking, CRC Press.However it is generally preferred to use recombinant manipulations tomake the in-frame fusion protein.

As noted, a fusion molecule or a conjugate molecule in accord with theinvention can be organized in several ways. In an exemplaryconfiguration, the C-terminus of the biologically active polypeptide isoperatively linked to the N-terminus of the effector molecule. Thatlinkage can be achieved by recombinant methods if desired. However, inanother configuration, the N-terminus of the biologically activepolypeptide is linked to the C-terminus of the effector molecule.

Alternatively, or in addition, one or more additional effector moleculescan be inserted into the biologically active polypeptide or conjugatecomplexes as needed.

Vectors and Expression

A number of strategies can be employed to express ALT-803. For example,a construct encoding ALT-803 can be incorporated into a suitable vectorusing restriction enzymes to make cuts in the vector for insertion ofthe construct followed by ligation. The vector containing the geneconstruct is then introduced into a suitable host for expression of thefusion protein. See, generally, Sambrook et al., supra. Selection ofsuitable vectors can be made empirically based on factors relating tothe cloning protocol. For example, the vector should be compatible with,and have the proper replicon for the host that is being employed. Thevector must be able to accommodate the DNA sequence coding for thefusion protein complex that is to be expressed. Suitable host cellsinclude eukaryotic and prokaryotic cells, preferably those cells thatcan be easily transformed and exhibit rapid growth in culture medium.Specifically preferred hosts cells include prokaryotes such as E. coli,Bacillus subtillus, etc. and eukaryotes such as animal cells and yeaststrains, e.g., S. cerevisiae. Mammalian cells are generally preferred,particularly J558, NSO, SP2-O or CHO. Other suitable hosts include,e.g., insect cells such as Sf9. Conventional culturing conditions areemployed. See, Sambrook, supra. Stable transformed or transfected celllines can then be selected. Cells expressing a fusion protein complex ofthe invention can be determined by known procedures. For example,expression of a fusion protein complex linked to an immunoglobulin canbe determined by an ELISA specific for the linked immunoglobulin and/orby immunoblotting. Other methods for detecting expression of fusionproteins comprising biologically active polypeptides linked to IL-15 orIL-15Rα domains are disclosed in the Examples.

As mentioned generally above, a host cell can be used for preparativepurposes to propagate nucleic acid encoding a desired fusion protein.Thus, a host cell can include a prokaryotic or eukaryotic cell in whichproduction of the fusion protein is specifically intended. Thus hostcells specifically include yeast, fly, worm, plant, frog, mammaliancells and organs that are capable of propagating nucleic acid encodingthe fusion. Non-limiting examples of mammalian cell lines which can beused include CHO dhfr-cells (Urlaub and Chasm, Proc. Natl. Acad. Sci.USA, 77:4216 (1980)), 293 cells (Graham et al., J Gen. Virol., 36:59(1977)) or myeloma cells like SP2 or NSO (Galfre and Milstein, Meth.Enzymol., 73(B):3 (1981)).

Host cells capable of propagating nucleic acid encoding a desired fusionprotein comples encompass non-mammalian eukaryotic cells as well,including insect (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S.pombe, P. pastoris., K. lactis, H. polymorpha; as generally reviewed byFleer, R., Current Opinion in Biotechnology, 3(5):486496 (1992)), fungaland plant cells. Also contemplated are certain prokaryotes such as E.coli and Bacillus.

Nucleic acid encoding a desired fusion protein can be introduced into ahost cell by standard techniques for transfecting cells. The term“transfecting” or “transfection” is intended to encompass allconventional techniques for introducing nucleic acid into host cells,including calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation, microinjection, viraltransduction and/or integration. Suitable methods for transfecting hostcells can be found in Sambrook et al. supra, and other laboratorytextbooks.

Various promoters (transcriptional initiation regulatory region) may beused according to the invention. The selection of the appropriatepromoter is dependent upon the proposed expression host. Promoters fromheterologous sources may be used as long as they are functional in thechosen host.

Promoter selection is also dependent upon the desired efficiency andlevel of peptide or protein production. Inducible promoters such as tacare often employed in order to dramatically increase the level ofprotein expression in E. coli. Overexpression of proteins may be harmfulto the host cells. Consequently, host cell growth may be limited. Theuse of inducible promoter systems allows the host cells to be cultivatedto acceptable densities prior to induction of gene expression, therebyfacilitating higher product yields.

Various signal sequences may be used according to the invention. Asignal sequence which is homologous to the biologically activepolypeptide coding sequence may be used. Alternatively, a signalsequence which has been selected or designed for efficient secretion andprocessing in the expression host may also be used. For example,suitable signal sequence/host cell pairs include the B. subtilis sacBsignal sequence for secretion in B. subtilis, and the Saccharomycescerevisiae α-mating factor or P. pastoris acid phosphatase phoI signalsequences for P. pastoris secretion. The signal sequence may be joineddirectly through the sequence encoding the signal peptidase cleavagesite to the protein coding sequence, or through a short nucleotidebridge consisting of usually fewer than ten codons, where the bridgeensures correct reading frame of the downstream protein sequence.

Elements for enhancing transcription and translation have beenidentified for eukaryotic protein expression systems. For example,positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp oneither side of a heterologous promoter may elevate transcriptionallevels by 10- to 400-fold in plant cells. The expression constructshould also include the appropriate translational initiation sequences.Modification of the expression construct to include a Kozak consensussequence for proper translational initiation may increase the level oftranslation by 10 fold.

A selective marker is often employed, which may be part of theexpression construct or separate from it (e.g., carried by theexpression vector), so that the marker may integrate at a site differentfrom the gene of interest. Examples include markers that conferresistance to antibiotics (e.g., bla confers resistance to ampicillinfor E. coli host cells, nptII confers kanamycin resistance to a widevariety of prokaryotic and eukaryotic cells) or that permit the host togrow on minimal medium (e.g., HIS4 enables P. pastoris or His-S.cerevisiae to grow in the absence of histidine). The selectable markerhas its own transcriptional and translational initiation and terminationregulatory regions to allow for independent expression of the marker. Ifantibiotic resistance is employed as a marker, the concentration of theantibiotic for selection will vary depending upon the antibiotic,generally ranging from 10 to 600 μg of the antibiotic/mL of medium.

The expression construct is assembled by employing known recombinant DNAtechniques (Sambrook et al., 1989; Ausubel et al., 1999). Restrictionenzyme digestion and ligation are the basic steps employed to join twofragments of DNA. The ends of the DNA fragment may require modificationprior to ligation, and this may be accomplished by filling in overhangs,deleting terminal portions of the fragment(s) with nucleases (e.g.,ExoIII), site directed mutagenesis, or by adding new base pairs by PCR.Polylinkers and adaptors may be employed to facilitate joining ofselected fragments. The expression construct is typically assembled instages employing rounds of restriction, ligation, and transformation ofE. coli. Numerous cloning vectors suitable for construction of theexpression construct are known in the art (λZAP and pBLUESCRIPT SK-1,Stratagene, La Jolla, CA, pET, Novagen Inc., Madison, WI, cited inAusubel et al., 1999) and the particular choice is not critical to theinvention. The selection of cloning vector will be influenced by thegene transfer system selected for introduction of the expressionconstruct into the host cell. At the end of each stage, the resultingconstruct may be analyzed by restriction, DNA sequence, hybridizationand PCR analyses.

The expression construct may be transformed into the host as the cloningvector construct, either linear or circular, or may be removed from thecloning vector and used as is or introduced onto a delivery vector. Thedelivery vector facilitates the introduction and maintenance of theexpression construct in the selected host cell type. The expressionconstruct is introduced into the host cells by any of a number of knowngene transfer systems (e.g., natural competence, chemically mediatedtransformation, protoplast transformation, electroporation, biolistictransformation, transfection, or conjugation) (Ausubel et al., 1999;Sambrook et al., 1989). The gene transfer system selected depends uponthe host cells and vector systems used.

For instance, the expression construct can be introduced into S.cerevisiae cells by protoplast transformation or electroporation.Electroporation of S. cerevisiae is readily accomplished, and yieldstransformation efficiencies comparable to spheroplast transformation.

The present invention further provides a production process forisolating a fusion protein of interest. In the process, a host cell(e.g., a yeast, fungus, insect, bacterial or animal cell), into whichhas been introduced a nucleic acid encoding the protein of the interestoperatively linked to a regulatory sequence, is grown at productionscale in a culture medium to stimulate transcription of the nucleotidessequence encoding the fusion protein of interest. Subsequently, thefusion protein of interest is isolated from harvested host cells or fromthe culture medium. Standard protein purification techniques can be usedto isolate the protein of interest from the medium or from the harvestedcells. In particular, the purification techniques can be used to expressand purify a desired fusion protein on a large-scale (i.e. in at leastmilligram quantities) from a variety of implementations including rollerbottles, spinner flasks, tissue culture plates, bioreactor, or afermentor.

An expressed protein fusion complex can be isolated and purified byknown methods. Typically the culture medium is centrifuged or filteredand then the supernatant is purified by affinity or immunoaffinitychromatography, e.g. Protein-A or Protein-G affinity chromatography oran immunoaffinity protocol comprising use of monoclonal antibodies thatbind the expressed fusion complex. The fusion proteins of the presentinvention can be separated and purified by appropriate combination ofknown techniques. These methods include, for example, methods utilizingsolubility such as salt precipitation and solvent precipitation, methodsutilizing the difference in molecular weight such as dialysis,ultra-filtration, gel-filtration, and SDS-polyacrylamide gelelectrophoresis, methods utilizing a difference in electrical chargesuch as ion-exchange column chromatography, methods utilizing specificaffinity such as affinity chromatography, methods utilizing a differencein hydrophobicity such as reverse-phase high performance liquidchromatography and methods utilizing a difference in isoelectric point,such as isoelectric focusing electrophoresis, metal affinity columnssuch as Ni-NTA. See generally Sambrook et al. and Ausubel et al. suprafor disclosure relating to these methods.

It is preferred that the fusion proteins of the present invention besubstantially pure. That is, the fusion proteins have been isolated fromcell substituents that naturally accompany it so that the fusionproteins are present preferably in at least 80% or 90% to 95%homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity(w/w) are most preferred for many pharmaceutical, clinical and researchapplications. Once substantially purified the fusion protein should besubstantially free of contaminants for therapeutic applications. Oncepurified partially or to substantial purity, the soluble fusion proteinscan be used therapeutically, or in performing in vitro or in vivo assaysas disclosed herein. Substantial purity can be determined by a varietyof standard techniques such as chromatography and gel electrophoresis.

The present fusion protein complexes are suitable for in vitro or invivo use with a variety of cells that are cancerous or are infected orthat may become infected by one or more diseases.

Human interleukin-15 (hIL-15) is trans-presented to immune effectorcells by the human IL-15 receptor a chain (hIL-15Rα) expressed onantigen presenting cells. IL-15Rα binds hIL-15 with high affinity (38pM) primarily through the extracellular sushi domain (IL-15RαSu). Asdescribed herein, the IL-15 and IL-15RαSu domains can be used togenerate a soluble complex (e.g., ALT-803) or as a scaffold to constructmulti-domain fusion complexes.

IgG domains, particularly the Fc fragment, have been used successfullyas dimeric scaffolds for a number of therapeutic molecules includingapproved biologic drugs. For example, etanercept is a dimer of solublehuman p75 tumor necrosis factor-α (TNF-α) receptor (sTNFR) linked to theFc domain of human IgG1. This dimerization allows etanercept to be up to1,000 times more potent at inhibiting TNF-α activity than the monomericsTNFR and provides the fusion with a five-fold longer serum half-lifethan the monomeric form. As a result, etanercept is effective atneutralization of the pro-inflammatory activity of TNF-α in vivo andimproving patient outcomes for a number of different autoimmuneindications.

In addition to its dimerization activity, the Fc fragment also providescytotoxic effector functions through the complement activation andinteraction with Fcγ receptors displayed on natural killer (NK) cells,neutrophils, monocyte cells, phagocytes and dendritic cells. In thecontext of anti-cancer therapeutic antibodies and other antibodydomain-Fc fusion proteins, these activities likely play an importantrole in efficacy observed in animal tumor models and in cancer patients.However these cytotoxic effector responses may not be sufficient in anumber of therapeutic applications. Thus, there has been considerableinterest in improving and expanding on the effector activity of the Fcdomain and developing other means of increasing the activity orrecruitment of cytolytic immune responses, including NK cells and Tcells at the disease site via immunotherapeutic molecules.

In an effort to develop human-derived immunostimulatory therapeutic,human IL-15 (hIL-15) and IL-15 receptor domains were used. hIL-15 is amember of the small four α-helix bundle family of cytokines thatassociates with the hIL-15 receptor α-chain (hIL-15Rα) with a highbinding affinity (Equilibrium dissociation constant (KD) ˜10⁻¹¹ M). Theresulting complex is then trans-presented to the human IL-2/15 receptorβ/common γ chain (hIL-15RβγC) complexes displayed on the surface of Tcells and NK cells. This cytokine/receptor interaction results inexpansion and activation of effector T cells and NK cells, which play animportant role in eradicating virally infected and malignant cells.Normally, hIL-15 and hIL-15Rα are co-produced in dendritic cells to formcomplexes intracellularly that are subsequently secreted and displayedas heterodimeric molecules on cell surfaces. Thus, the characteristicsof hIL-15 and hIL-15Rα interactions suggest that these inter chainbinding domains could serve as a human-derived immunostimulatory complexand as a scaffold to make soluble dimeric molecules capable oftarget-specific binding.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

Example 1: Induction of Lymphocyte Proliferation and Activation byALT-803

Human blood buffy coats from normal individuals were used to isolateperipheral blood mononuclear cells (PBMCs) with Histopaque-1077. PBMCswere cultured at 37° C. with 5% CO₂ in RPMI-10 medium (RPMI-1640,2-mercaptoethanol, penicillin-streptomycin-glutamine, non-essentialamino acids, sodium pyruvate, and 10% fetal bovine serum) with thevarious amounts of ALT-803. By “ALT-803” is meant a complex comprisingIL-15N72D noncovalently associated with a dimeric IL-15RαSu/Fc fusionprotein, wherein said complex exhibits immune stimulating activity.Optionally, the IL-15N72D and/or IL-15RαSu/Fc fusion protein comprisesone, two, three, four or more amino acid variations relative to areference sequence. An exemplary EL-15N72D amino acid sequence isprovided below. (See, e.g., U.S. Ser. No. 13/769,179, incorporatedherein by reference).

An exemplary EL-15N72D nucleic acid sequence is provided below (withleader peptide) (SEQ ID NO: 1):

(Leader peptide) atggagacagacacactcctgttatgggtactgctgctctgggttccaggttccaccggt- (IL-15N72D)aactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacgacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct (Stop codon) taa

An exemplary EL-15N72D amino acid sequence is provided below (withleader peptide) (SEQ ID NO: 2):

(Leader peptide) METDTLLLWVLLLWVPGSTG- (IL-15N72D)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the mature EL-15N72Dpolypeptide (SEQ ID NO: 3):

(IL-15N72D) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTS

An exemplary EL-15RαSu/Fc nucleic acid sequence (with leader peptide) isprovided below (SEQ ID NO: 4):

(Leader peptide) atggacagacttacttcttcattcctgctcctgattgtccctgcgtacgtcttgtcc- (IL-15RαSu) atcacgtgccctccccccatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccagggagcggtacatttgtaactctggtttcaagcgtaaagccggcacgtccagcctgacggagtgcgtgttgaacaaggccacgaatgtcgcccactggacaacccccagtctcaaatgtattaga-(IgG1 CH2-CH3 (Fc domain))gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctettccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc tccgggtaaa-(Stop codon) taa

An exemplary IL-15RαSu/Fc amino acid sequence (with leader peptide) isprovided below (SEQ ID NO: 5):

(Leader peptide) MDRLTSSFLLLIVPAYVLS- (IL-15RαSu)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR-(IgG1 CH2-CH3 (Fc domain))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some cases, the mature IL-15RαSu/Fc protein lacks the leader sequence(SEQ ID NO: 6):

(IL-15RαSu) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR- (IgG1 CH2-CH3 (Fc domain))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

To assess lymphocyte subset proliferation and activation,ALT-803-treated cells were stained with either a combination ofBrilliant Violet 510-anti-CD4, PECY7-anti-CD8, and Brilliant Violet421-anti-CD16 antibodies for surface markers, followed by intracellularstaining with FITC-anti-granzyme B antibody or a combination ofBrilliant Violet-anti-CD4, PE-anti-CD8, PECY7-anti-CD335,PerCP-CY5.5-anti-CD69 and APC-anti-CD25 antibodies for surface markers,followed by intracellular staining with FITC-anti-perforin antibody. Forintracellular staining, the cells were fixed with fixation buffer (PBSwith 2% paraformaldehyde) and incubated at room temperature for 20minutes. The fixed cells were permeabilized in Permeabilization Buffer(PBS with 0.1% saponin and 0.05% sodium azide) and stained withFITC-labeled antibodies specific to human granzyme B or perforin. Thepercentage of CD4⁺ T cells, CD8⁺ T cells and CD16⁺ NK cells in theALT-803-activated PBMCs, and the expression of CD25 and CD69 activationmarkers on CD4⁺ T cells, CD8⁺ T cells and CD335⁺ NK cells were analyzedon a FACSVerse flow cytometer using FACSuite software.

There was no significant change in the percentage of CD4⁺ T cells, CD8⁺T cells or NK cells in the human PBMCs from different donors following 5days of incubation in medium containing 0.01 to 10 nM ALT-803 comparedwith medium control (FIG. 1A and FIG. 1B). Since previous studies haveshown that ALT-803 can stimulate proliferative responses of human PMBCsin vitro, the incubation time of the culture was extended to increasethe sensitivity of this assay. As shown in FIG. 1C (Donor-A) and FIG. 1D(Donor-B), after 7 days of culture in the presence of 10 nM ALT-803, thepercentage of CD4⁺ T cells decreased and the percentage of CD8⁺ T cellsincreased in PBMC cultures when compared with that observed in cellsincubated in the absence of ALT-803. This finding was more apparent whenthe CD4/CD8 ratio was analyzed (FIG. 1E: Donor-A and FIG. 1F: Donor-B).There was no significant difference in the percentage of NK cells inPBMC cultures incubated up to 10 days in the presence or absence ofALT-803 (FIGS. 1C & D).

The in vitro effects of ALT-803 were compared to IL-15 on proliferativeof human immune cell subsets from different donors. Addition of either0.5 nM ALT-803 or 0.5 nM IL-15 to human PBMC cultures resulted in˜2-fold increase in lymphocyte counts after a 7-day incubation period.ALT-803 was as potent as IL-15 in increasing the absolute number of CD8⁺T-cell and NK cell subsets (FIG. 2A and FIG. 2B). ALT-803 alsosignificantly increased the absolute counts of CD4⁺ T cells whereasIL-15 increased the absolute counts of Treg cells. In addition, theexpression of activation markers, CD25 and CD69, on the immune cellsubsets was examined in order to understand immunostimulatory effects ofALT-803. As shown in FIG. 3A-FIG. 3D, in vitro treatment with ALT-803was capable of increasing CD25 expression by CD4⁺ T cells, CD8⁺ T cellsand NK cells in a concentration dependent manner. Notably, CD25upregulation by ALT-803 was most significant on CD4⁺ T cells compared tothat seen on CD8⁺ T or NK cells. CD25 expression by CD8⁺ T cells wasminimally induced by ALT-803. In contrast to CD25, CD69 expression washighly upregulated on NK cells by ALT-803 incubation, whereas little orno treatment effects were seen on CD69 expression by CD4⁺ T cells.

The studies were also conducted to assess whether ALT-803 could induceNK cells and CD8⁺ T cells to express higher levels of granzyme andperforin, which play a key role in cytolytic responses. Human PBMCs wereactivated in vitro with ALT-803 as described above and followed by Abstaining to differentiate CD8⁺ T cell and NK cell subsets and thenintracellularly stained with FITC-labeled antibodies specific to humangranzyme B or perforin. As shown in FIG. 4A-FIG. 4D, ALT-803 was capableof upregulating the expression of granzyme B and perforin by CD8⁺ Tcells (FIG. 4A and FIG. 4C) and NK cells (FIG. 4B and FIG. 4D) in aconcentration dependent manner. Moreover, ALT-803-mediated induction ofgranzyme B expression in both CD8⁺ T cells and NK cells was moresignificant than ALT-803-mediated effects on perforin expression. Thesefindings are consistent with the notion that ALT-803-induced expressionof granzyme B and/or perforin may play a role to enhanced cytotoxicityof PBMCs following incubation with ALT-803.

Additional studies were conducted to compare the effects of ALT-803 onhuman and mouse immune cells. Previous studies have shown that theimmunostimulatory effects of proteins on human PBMCs could varysignificantly depending on whether the protein is present in an aqueousor immobilized form. Thus, cytokine release and proliferation assayswere conducted on human and mouse cells using ALT-803 as soluble proteinor as plastic-immobilized wet or air-dried protein prepared. ALT-803 wastested at 0.08, 0.8 and 44 nM, which correspond to maximal serumconcentrations in human of a 0.3, 3.0 and 170 μg/kg i.v. dose,respectively. For proliferation assays, human PBMCs and mouse CD3⁺ cellsenriched from splenocytes (CD3+ T Cell Enrichment column, R&D System)were labeled with Celltrace™ Violet (Invitrogen) and cultured in wellscontaining PBS or ALT-803. As a positive control, 27 nM of anti-CD3 Ab(145-2C11 for mouse splenocytes and OKT3 for human PBMCs) was added toseparate wells in the same assay formats. The cells were incubated for 4days and then analyzed by flow cytometry to determine cell proliferationbased on violet dye dilution. Additionally, human and mouse immune cellswere cultured as described above for 24 and 48 h and cytokines releasedinto the media were measured using Human and Mouse Th1/Th2/Th17Cytometric Bead Array Cytokine kits according to manufacturer'sinstructions (BD Biosciences). For immune cell subset analysis, humanPBMCs were cultured in various concentrations of ALT-803 or IL-15 andstained with antibodies specific to CD4, CD8, CD335, CD16 or CD19 orwith a human Treg kit (BioLegend). In some experiments, cells were alsostained with antibodies specific to CD69, granzyme B or perforin asdescribed above.

Incubation with immobilized ALT-803 for one day or four days (FIG. 5A)resulted in elevated IFN-γ release by human PBMCs. Soluble IL-6 was alsoincreased in 4-day human PBMC cultures treated with ALT-803, though thiseffect was not dose dependent (FIG. 5A). In contrast, ALT-803 had noeffect on TNF-α, IL-4, IL-10, or IL-17A release by 4-day human PBMCcultures. When tested in parallel cultures, a positive control anti-CD3mAb induced release of IFN-7, TNF-α, IL-10, IL-4 and IL-17A. Compared tohuman immune cells, mouse splenocytes exhibited a similar, but lessintense response for IFN-γ release following incubation with ALT-803(FIG. 5B). ALT-803 also induced TNF-α production from mouse splenocytes,but showed no significant effect on levels of IL-6, IL-2, IL-10, IL-4and IL-17A. Conversely, murine lymphocytes incubated with immobilizedanti-CD3 antibody showed significantly elevated release of all of thecytokines tested. Together, these findings indicate that ALT-803primarily stimulates IFN-γ production by human and mouse immune cells,in contrast to the broad profile of cytokines induced by anti-CD3antibodies.

The ability of ALT-803 to induce in vitro proliferation of CellTrace™Violet labeled human and mouse immune cells was also evaluated.Pronounced proliferation of mouse lymphocytes was evident followingincubation with 0.7 nM to 44 nM soluble ALT-803 (FIG. 5C). Up to 83% ofthe cells in the high-dose soluble ALT-803 group underwent one to sixrounds of cell division during the 4-day incubation period. Little or noproliferation was detected in untreated murine cells or those treatedwith 0.07 nM soluble ALT-803. As expected, murine lymphocytes incubatedwith immobilized anti-CD3 antibody exhibited strong proliferativeresponses. ALT-803 dose-dependent lymphocyte proliferation was alsoobserved in human PBMC cultures, but this response was considerably lessthan that seen for mouse cells. Overall, less than 20% of all humanlymphocytes proliferated in response to high-dose ALT-803 and theseresponses were less than those induced by the positive control anti-CD3antibody. Additionally, individual variations in cell proliferativeresponses to both ALT-803 and anti-CD3 Ab were observed in the bloodlymphocytes of different donors.

Example 2: Induction of Cell-Mediated Cytotoxicity by ALT-803 andALT-803 in Combination with Antibodies

To assess if ALT-803 affected cell-mediated cytotoxicity, isolated humanPBMCs from blood buffy coats were used as effector cells. Daudi humanB-cell lymphoma cells and K562 human myelogenous leukemia cells wereused as target cells and labeled with Celltrace Violet at 5 μM inRPMI-10 at 37° C. for 20 minutes as described by the manufacturer. Theeffector cells were mixed with the violet-labeled target cells andincubated at 37° C. with 5% CO₂ in RPMI-10 with and without ALT-803 forthe indicated times. In some experiments, anti-CD20 Ab (Rituximab, 10nM) specific to CD20 expressed on the surface of Daudi cells was addedto the effector:Daudi cell culture to determine the effects of ALT-803on anti-CD20 Ab-mediated antibody-dependent cellular cytotoxicity(ADCC). The mixtures of effector cells and target cells were harvestedby centrifugation and resuspended in RPMI-10 without phenol red with 2μg/ml of propidium iodide. Cytotoxicity of the effector cells againstthe target cells was evaluated by flow cytometry by determining thepercentage of dead violet-labeled target cells after propidium iodidepositive staining.

As shown in FIG. 6A-FIG. 6D, fresh human PBMCs had weak cytotoxicityagainst Daudi and K562 cells in the absence of ALT-803. In contrast,PBMCs had very strong cytotoxicity against Daudi and K562 tumor targetcells in the presence of ALT-803 at 10 nM. Daudi tumor cells expressCD20 molecules, which can be recognized by the anti-CD20 Ab (Rituximab).As indicated herein, CD20 is an established target for therapeuticantibody treatment of hematologic tumors and autoimmune diseases. HumanPBMCs are capable of lysing Daudi cells by means of ADCC mediated byRituximab alone (FIG. 6C, Medium control). Interestingly, ALT-803 wasalso capable of significantly augmenting Rituximab-mediated ADCCactivity of human PBMCs against Daudi cells. As shown in FIG. 6D, a timedependent increase in ALT-803-mediated effects on PBMC cytotoxicity andADCC was observed, with little or no responses seen after one day ofALT-803 incubation and elevated target cell killing observed with eachadditional day of incubation.

Additionally, ALT-803 concentration dependent induction of human PBMCcytotoxicity against K562 and Daudi cells was investigated. Fresh humanPBMCs were mixed with Celltrace Violet labeled K562 cells or Daudi cellsin RPMI-10 medium with various concentrations (from 0.01 nM to 10 nM) ofALT-803 followed by incubation for 3 days. The cytotoxicity of the humanPBMCs against the target cells was evaluated by flow cytometry asdescribed above. Consistent with the results in FIG. 6A-FIG. 6D, ALT-803was capable of augmenting cytotoxicity of human PBMCs against Daudi andK562 cells at 10 nM (FIG. 7A and FIG. 7B: Donor-A & Donor-B). Moreover,ALT-803 at as low as 0.01 nM also demonstrated increased cytotoxicity ofhuman PBMCs against the target cells. The PBMCs from two individualsexhibited significantly different cytotoxic activities against thedifferent tumor target cells. PBMCs from Donor-A (FIG. 7A) exhibitedmuch higher baseline and ALT-803-induced cytotoxicity against K562 thanDaudi cells. In contrast, PBMCs from Donor-B (FIG. 7B) exhibited similarbaseline and ALT-803-induced cytotoxicity against the two differenttarget cells. This finding may be important in understanding thepotential variability of ALT-803-mediated clinical responses indifferent patients. Similar cytotoxicity assays are incorporated as acorollary assessment of patients' immune responses to ALT-803 as part ofthe clinical use of this molecule.

The concentration-dependent effects of ALT-803 on tumor specific ADCCwere also further assessed. As shown in FIG. 8A-FIG. 8B, ALT-803 atconcentrations at as low as 0.01 nM was capable of augmenting the ADCCactivity of anti-CD20 mAb (10 nM) against human Daudi B lymphoma cells.This response was observed with human PMBC effector cells incubated withDaudi cells at a 2:1 E:T ratio, indicating the sensitivity of thisactivity. In order to identify the immune effector responsible for theobserved target tumor cell killing, NK cells were isolated from humanPBMCs by MACS and used as the effector cells in the above ADCC assay.Similarly to results with total human PBMC, NK cells were capable ofkilling Daudi cells via Rituximab-mediated ADCC activity that wasenhanced by addition of ALT-803 (FIG. 8C). In contrast, NK cell-depletedPBMCs (non-NK cell) did not display Daudi cell killing detectable ADCCactivity in this setting (FIG. 8D). Moreover, addition of a controlantibody, HOAT (humanized anti-human tissue factor IgG1 Ab), to NK cellsdid not provide detectable ADCC activity against Daudi cells with orwithout addition of ALT-803.

The ability of ALT-803 to augment ADCC activity of mouse immune cellsagainst human Daudi cells was also examined. In this study, SCID micewere inoculated with Daudi cells (10×10⁶ per mouse) on study day 0 (SD0)and treated on SD15 and SD18 with ALT-803 (0.2 mg/kg), Rituximab (10mg/kg), or ALT-803 (0.2 mg/kg)+Rituximab (10 mg/kg). The mice weresacrificed 4 days post the second treatment and splenocytes wereprepared. Thus, the splenocytes may have different activation states asa result of the in vivo treatment. The splenocytes were then mixed withDaudi target cells at E:T ratio 20:1 in RPMI-10 medium alone or mediumwith Rituximab (10 nM), ALT-803 (10 nM) or Rituximab (10 nM)+ALT-803 (10nM). After 2 days of incubation at 37° C., Daudi target cell viabilitywas assessed by analysis of propidium iodide staining of violet-labeledDaudi cells on a BD FACSVerse. As shown in FIG. 9A, addition of ALT-803to splenocytes derived from control treated mice was capable ofaugmenting the anti-CD20 mAb directed ADCC against human Daudi cells.Additionally, in vivo stimulation with ALT-803 resulted in splenocytesthat were more active in anti-CD20 mAb directed ADCC against human Daudicells. These results are consistent with the finding using human immunecells indicating that ALT-803 treatment can potentiate tumor-specificADCC responses.

The capacity of ALT-803 to augment ADCC activity of immune cells againstother human tumor cells was examined. HER-2 is an established target fortherapeutic antibody treatment of solid tumors, including breast cancerand gastric or gastroesophageal junction adenocarcinoma. To assess theeffects of ALT-803 on ADCC activity against HER-2-positive tumors, theSK-BR-3 human breast cancer cell line that over-expresses HER-2 was usedas a target cell line and an anti-human HER-2 antibody (clone 24D2) wasused as a tumor cell-targeted Ab. To generate activated effector cells,Balb/c mice were injected with 0.2 mg/kg ALT-803 intravenously on studyday (SD) 0. On SD3, mice were sacrificed and spleens were harvested.Activated splenocytes were mixed with CellTrace Violet-labeled SK-BR-3cells in 10:1 E:T ratio. Cells were co-cultured at 37° C. in RIO mediacontaining various concentrations of anti-HER2 antibody (clone 24D2),ALT-803 or both agents. After 24 hrs, cell mixtures were collected anddead cells were stained with propidium iodide. The percentage of deadSK-BR-3 cells was examined using flow cytometry. As shown in FIGS. 9B,incubation of SK-BR-3 cells with activated splenocytes in the presenceof either anti-HER2 Ab or ALT-803 alone did not result in SK-BR-3 celldeath compared to media controls. However, combined treatment of ALT-803(at 0.01-1.0 nM) with anti-HER2 antibody (clone 24D2) (at 0.1 to 10 nM)significantly increased ADCC activity of the splenic immune cellsagainst SK-BR-3 human breast cancer cells. These results verify thatALT-803 can augment ADCC responses to several different therapeuticallyestablished disease targets.

Example 3: Antitumor Activity of ALT-803+Tumor-Specific AntibodyTreatment in Tumor-Bearing Mice

The ability of ALT-803 to augment the antitumor activity of anti-CD20mAb was further evaluated in SCID mice bearing Daudi tumors. These micehave functional NK cells which likely mediate ADCC responses againsttumors. For this study, Fox Chase SCID female mice (Harlan,C.B-17/IcrHsd-Prkdc-scid: 6 weeks-old) were inoculated intravenously(i.v.) with Daudi cells (10×10⁶ per mouse) (3 mice/group). Thetumor-bearing mice were treated i.v. with PBS, ALT-803 (0.2 mg/kg),Rituximab (10 mg/kg) or ALT-803 (0.2 mg/kg)+Rituximab (10 mg/kg) 15 dayspost tumor inoculation and three days later. Four days after secondtreatment the mice were sacrificed and the levels of the Daudi cells inbone marrow were determined. The percentage of Daudi cells of femur bonemarrow cells was assessed following staining with PE-conjugatedanti-human HLA-DR antibody (Biolegend) and flow cytometry analysis. Theresults shown in FIG. 10 indicate that ALT-803 and anti-CD20 mAbmonotherapies are capable of reducing the percentage of Daudi cells inthe bone marrow of tumor-bearing mice. However the combination ofALT-803 plus anti-CD20 mAb provided the greatest antitumor activityreducing bone marrow Daudi cells from 38% in control mice to 5% inALT-803+Rituximab treated mice. Dose responses studies in this modelconfirmed that as little as 0.02 mg/kg ALT-803 was capable of augmentingthe antitumor activity of anti-CD20 mAb (FIG. 11 ).

The ability of ALT-803 plus anti-CD20 mAb to improve survival of micebearing Daudi cell tumors was also evaluated. For this study, Fox ChaseSCID female mice were inoculated intravenously (i.v.) with Daudi cells(10×10⁶ per mouse). The tumor-bearing mice were treated i.v. with PBS,ALT-803 (suboptimal 0.05 mg/kg), Rituximab (10 mg/kg) or ALT-803 (0.05mg/kg)+Rituximab (10 mg/kg) 15 days post tumor inoculation and threedays later. Survival (including morbidity based on hind leg paralysis)of the mice was monitored. As shown in FIG. 12 , tumor bearing mice ofthe PBS, suboptimal ALT-803 and Rituximab monotherapy groups showedmedian survival of 26-30 days whereas the mice treated with suboptimalALT-803 plus Rituximab had a mean survival of greater than 50 days,indicating combined therapy significantly prolongs survival of micebearing B cell lymphomas. Together, these results confirm thesynergistic antitumor effects of ALT-803 in combination withtumor-specific antibodies in vivo. It is also noteworthy that thecombination of ALT-803 plus anti-CD20 mAb did not cause any significantsigns of toxicity in the tumor-bearing animals for studies describedabove, which indicates that these combinations are well tolerated.

Example 4: Antitumor Activity of ALT-803 in Combination with ImmuneCheckpoint Blockers in Tumor-Bearing Mice

Immune checkpoint blockers including antibodies against CTLA-4, PD-1,PD-L1, PD-L2, B7-H3, B7-H4, LAG-3, BTLA, TIM-3, VISTA, IDO, A2aR, HVEM,KIRs, NKG2A, NKG2D, CEACAM-1, 2B4, CD200R, and their ligands and othertargets described herein may be capable of promoting immune responses byinhibiting immune suppressive signals. As indicated above, ALT-803 is animmunostimulatory molecule that promotes proliferation and activity ofNK cells and T cells. However, its effectiveness may be limited byinhibitory checkpoints and pathways that can attenuate immune responses.Strategies that abrogate these negative regulators and enhance theactivity of ALT-803 could provide therapeutic benefit.

To examine the antitumor activity of ALT-803 in combination withblockade of the CTLA-4-CD80/CD86 pathway, a lung metastasis model wasdeveloped using BALB/c mice injected i.v. with the CT26 murine coloncarcinoma cell line. Groups of 4-6 mice were injected i.v. with 2×10⁵CT26 tumor cells on day 0 (SD0). In the ALT-803 groups, each mousereceived 4 μg of i.v. ALT-803 twice a week for two weeks starting onSD1. Along with ALT-803, some mice received of either anti-PD-L1antibody (Ab) (clone 9G2), anti-CTLA-4 (clone UC10-4F10-11) Ab or bothat 100 μg per injection (per Ab) administered i.v. twice a week for 2weeks starting on SD1. In the recombinant human IL-15 groups, each mousereceived 5 μg of IL-15 intraperitoneally (i.p.) daily, 5 times a weekfor 2 weeks starting on SD1. Along with IL-15, animals also receivedi.v. treatment of both anti-PD-L1 Ab and anti-CTLA-4 Ab (100 μg perinjection) on SD1, SD4, SD8 and SD11. Control mice received injectionsof PBS. Mice were assessed daily for survival as an efficacy endpoint.

As shown in FIG. 13 , the median survival of BALB/c mice bearing CT26tumors was 15 to 20 days depending on the study. Treatment with ALT-803alone significantly prolonged survival of tumor bearing mice whencompared to the control group. Addition of anti-PD-L1 Ab treatment toALT-803 did not appear to improve survival in these mice. In contrast,the combination of ALT-803 and anti-CTLA4 Ab (with or without anti-PD-L1Ab) significantly increased the survival of CT26 tumor bearing micecompared to the PBS and ALT-803 groups. Previous studies in this tumormodel showed that anti-CTLA4 Ab monotherapy did not improved survival.Thus, the combination of ALT-803 and anti-CTLA4 Ab (but not anti-PD-L1Ab) acts synergistically in providing more efficacious antitumorresponses as measured by prolonged survival on tumor-bearing mice.

The effects of PD-1-PD-L1 blockade in combination with ALT803 werefurther evaluated in the 5T33P myeloma model in immunocompetent C57BL/6mice. Groups of 5 mice were injected i.v. with 1×10⁷ 5T33P tumor cellson SD0. Treatment began on SD4. In the IL-15 group, each mouse received5 μg of IL-15 i.v. on SD4 and SD11. Along with IL-15, some animals alsoreceived anti-PD-L1 Ab (clone 9G2, 200 μg/mouse/injection i.p.) oranti-CTLA-4 Ab (clone UC10-4F10-11, 200 μg/mouse/injection i.p.) once aweek on SD4 and SD11 respectively. In ALT-803 group, each mouse receivedtwo i.v. injections of suboptimal ALT-803 at 1 μg/mouse/injection on SD4and SD11. Along with ALT-803, some mice received either anti-PD-L1 Ab,anti-CTLA-4 Ab or both antibodies as indicated above. Additionally,groups of mice received anti-PD-L1 Ab or anti-CTLA-4 Ab monotherapy andreceived injections of PBS. Mice were assessed daily for survival and/orfull paralysis in both hind legs as the study endpoint. As shown in FIG.14A, the median survival of C57BL/6 mice bearing 5T33P tumors was 24days. Treatment with a suboptimal dose of ALT-803 did not significantlychange animal survival, but administration of suboptimal ALT-803 plusanti-PD-L1 Ab resulted in survival of all animals in this study groupfor at least 50 days. In contrast, the combination of ALT-803 andanti-CTLA4 Ab did not significantly prolong mouse survival.Additionally, when suboptimal levels of both ALT-803 and anti-PD-L1 Abwere tested as monotherapies and in combination, significantly longersurvival was observed in C57BL/6 mice bearing 5T33P tumors followingtreatment of ALT-803+anti-PD-L1 Ab compared to mice treated with eithermonotherapy (FIG. 14B). Thus, the combination of ALT-803 plus anti-PD-L1Ab provides synergistic antitumor activity in mice bearing myelomatumors.

To better understand the divergent activities of the ALT-803 combinationtherapies in these two models, the tumor cell lines were stained forligands of the PD1 and CTLA4 receptors. As shown in FIG. 15 , CT26 cellsexpress ligands for CTLA4 but not PD1. In contrast, 5T33P tumor cellsexpress PD-L1 but not ligands for CTLA4. These results are consistentwith the antitumor activities of anti-CTLA4 and anti-PD-L1 Abs incombination ALT-803 in each of these tumor models, indicating thatstaining tumors with ligands for immune check point receptors mayprovide a predictive indicator for response to ALT-803 plus immune checkpoint blockers.

Using an established model for glioblastoma described in Zeng et al.2013 Int J Radiat Oncol Biol Phys., 86:343-9, studies of ALT-803monotherapy and ALT-803 in combination with anti-PD-1 mAb were alsocarried out in C57BL/6 mice that had been intracranially implanted withthe glioblastoma cell line, GL261-luc. Treatment with multiple doses ofALT-803 (3 or 4 doses) or anti-PD-1 mAb (3 doses) as monotherapystarting 7 to 10 day post-tumor implantation exhibited similar increasesin antitumor activity and prolonged animal survival when compared toPBS-treated controls. The combination of ALT-803 and anti-PD1 mAbtreatment further extended median survival times of tumor-bearing mice.Additionally, anti-PD-1 mAb in combination with 4 doses of ALT-803increased the percentage of long-term tumor free survivors (>60 dayspost-implantation) to 40% from the 20% rate observed in mice treatedwith anti-PD-1 Ab and ALT-803 monotherapy. Interestingly, the “cured”mice were resistant to tumor rechallenge, suggesting treatment-inducedimmune memory response against the tumor. These results suggest thatcombining the immunostimulatory activity of ALT-803 with the checkpointblocker, anti-PD-1 Ab, has a beneficial effect in prolonging survival ofglioblastoma tumor bearing mice.

It is also noteworthy that the combination of ALT-803 plus checkpointinhibitor blockade did not cause any significant signs of toxicity inthe tumor-bearing animals for studies described above, which indicatesthat these combinations are well tolerated.

Example 5: Toxicity of ALT-803 in Mice

To evaluate the safety profile and therapeutic index of ALT-803 inanimals and estimate the safe and efficacy human dose, toxicity studiesof multidose ALT-803 treatment were conducted in mice and cynomolgusmonkeys. C57BL/6N mice (10 mice/sex/group) were administered 0.1, 1.0 or4.0 mg/kg ALT-803 or PBS via the tail vein weekly for 4 consecutiveweeks. Four days after the last injection (day 26), assessmentsincluding physical examination, blood chemistry, hematology, grossnecropsy, body and organ weight measurements and histopathology wereperformed (5 mice/sex/group). Similar assessments were performed on theremaining mice fourteen days after the last treatment (day 36). In asecond study, C57BL/6N mice (15 mice/sex/group) were treated with 4weekly i.v. injections of 0.1 or 1.0 mg/kg ALT-803 or PBS. Toxicityassessments as described above were performed four days (day 26) (10mice/sex/group) or 4 weeks after the last injection (day 50) (5mice/sex/group).

The safety and pharmacodynamic profiles of ALT-803 was assessed inhealthy C57BL/6N mice injected i.v. with 0.1, 1.0 or 4.0 mg/kg ALT-803or PBS weekly for four consecutive weeks. Mice receiving 4.0 mg/kgALT-803 exhibited signs of toxicity (i.e., weight loss, hair loss) andmortality between 4 to 20 days after treatment initiation. Post-mortemnecropsy did not determine the cause of death but observations (i.e.,pulmonary edema, enlarged spleens) were consistent with cytokine-inducedlethal inflammatory responses. Mortality was not observed in micetreated with 1.0 or 0.1 mg/kg ALT-803. Dose dependent increases inspleen weights and white blood cell (WBC) counts were seen 4 days afterthe last dose of ALT-803 (Day 26) (FIG. 16 ). Of the WBCs, absolutecounts for lymphocytes, neutrophils and monocytes each increased over 8fold in 1.0 mg/kg ALT-803-treated mice compared to controls. Two weeks(Day 36) and four weeks (Day 50) after treatment (FIG. 16A-FIG. 16F),neutrophil counts remained elevated in 1.0 mg/kg ALT-803-treated mice,but lymphocyte counts returned to control levels. Histopathologicalanalysis verified ALT-803 dose-dependent stimulation of immune cellproliferation and lymphocyte infiltration in the spleen, liver, thymus,kidney, lungs and lymph nodes on day 26 and to a lesser degree on day 36and day 50. The results of these studies defined the tolerable dose ofmultidose ALT-803 treatment at up to 1 mg/kg in mice.

Example 6: Toxicity. Pharmacodynamics (PD), and Pharmacokinetics (PK) ofALT-803 in Cynomolgus Monkeys

A study was performed under Good Laboratory Practice regulations toevaluate the effects of multidose i.v. administration of ALT-803 incynomolgus monkeys. Animals (5 monkeys/sex/group) were treated weeklyfor 4 consecutive weeks (days 1, 8, 15 and 22) with 0.03 or 0.1 mg/kgALT-803 or PBS administered as a ˜3 min i.v. injection. Throughout thein-life phase of the study, animals were assessed for clinical andbehavioral observations, food consumption, body weight, cardiac andocular function. Blood was taken for hematology, chemistry andcoagulation assessments (pre-dosing and days 5, 26 and 36 post-dosing)and for immune cell analysis. Serum was taken for immunogenicity testingand PK analyses conducted using qualified enzyme-linked immunosorbentassay (ELISA) methods. Urine was collected for urinalysis (pre-dosingand days 4, 25 and 35). Clinical pathology assessments includingphysical examination, gross necropsy, organ weight measurements andhistopathology were performed four days (day 26) (3 animals/sex/group)and 2 weeks after the last injection (day 36) (2 animals/sex/group).

Based on allometric scaling to the tolerable murine dose, the activityand toxicity profiles of multidose i.v. treatment of ALT-803 at 0.1 and0.03 mg/kg were assessed in healthy cynomolgus monkeys. PK analysisafter the first dose estimated the 1 elimination half-life of ALT-803 atapproximately 7.6 hrs, which did not appear to differ significantlybetween dose levels (FIG. 17 ). The C_(max) value of 30 nM for 0.1 mg/kgALT-803 is consistent with full recovery of the administered dose,whereas C_(max) and AUC_(INF) values indicate ˜30% less recovery at the0.03 mg/kg dose. However, even at the low dose level, the C_(max) of 6nM in the serum was over 50 higher than the 0.1 nM concentration foundto stimulate immune cell proliferation, activation and cytotoxicity invitro.

Monkeys receiving 4 consecutive weekly injections of ALT-803 showed adose-dependent reduction in appetite during the first 2 weeks of thetreatment period. However, there were no significant differences in meanbody weights or any other dose-related clinical or behavior observationsamong the groups during the study. Additionally, organ weights were notsignificantly different in ALT-803 treated animals compared to controls.

The most biologically relevant changes observed following weekly ALT-803treatment were dose-dependent increases in blood WBC and lymphocytecounts (FIG. 18A-FIG. 18H). At the end of the four week dosing period,absolute lymphocyte counts increased 1.5-fold in animals receiving 0.1mg/kg ALT-803 and then returned to control levels after a 2-weekrecovery period. Of the lymphocyte subsets, transient dose-dependentincreases in NK cell and CD4⁺ and CD8⁺ T cell counts were seen posttreatment (FIG. 18A-FIG. 18F). Blood monocyte counts also increased in0.1 mg/kg ALT-803 treated monkeys whereas blood neutrophil levels werenot different among the treatment groups. These results contrast withprevious studies of IL-15 administration to macaques and rhesus monkeyswhere the major toxicity reported was grade 3/4 transient neutropenia.

In addition to changes in blood immune cell levels, there wasdose-dependent increase in mild multifocal lymphocytic infiltration inthe livers, kidneys and lungs of ALT-803 treated monkeys based onhistopathology conducted 4 days after the last dose of ALT-803.Scattered mild liver necrosis was also seen with increased frequency inALT-803 treated animals. Clinical chemistry at this time point showed adecrease in serum albumin in the high-dose ALT-803 group compared tocontrols (0.1 mg/kg ALT-801, 3.85±0.12 g/dL; PBS, 4.46f0.13 g/dL;P<0.01), which may be a consequence of inflammatory responses in theliver. However, serum liver enzyme levels were not elevated in ALT-803treated animals compared to controls. Bone marrow hyperplasia wasobserved in most animals but with increased severity in the high-doseALT-803 group. Lesions in a majority of affected organs in the ALT-803treated groups were reduced in incidence and severity by two weekspost-treatment and were consistent with findings in the control animals.Generally, the ALT-803-mediated effects on blood and tissue lymphocytesobserved in this study are consistent with transient responses reportedfor non-human primates treated with IL-15 twice weekly at up to 0.1mg/kg or daily at 10 to 50 μg/kg.

Example 7: Comparative Studies of Intravenous and Subcutaneous ALT-803

Emerging data from ongoing trials using recombinant human IL-15(rhIL-15) product suggests that intravenous dosing is likely not optimalfor IL-15 because it induces a high Cmax and secondary cytokine release(IL-6 and IFN-7) that affects its tolerability and is thereforelimiting. Pre-clinical and clinical studies with IL-2 indicate thatsubcutaneous dosing is safer and provides much better tolerability. Forexample, Waldmann and colleagues conducted the first solid tumor trialof rhIL-15 in human using daily intravenous bolus infusion for 12consecutive days (Conlon et al., 2015. J. Clin. Oncol., 33: 74-82). Doselimiting toxicities observed in the 3.0 and 1.0 μg/kg per day cohortwere grade 3 hypotension, thrombocytopenia, and elevations of alaninetransaminase (ALT) and aspartate transaminase (AST). The maximumtolerated dose (MTD) was declared at 0.3 μg/kg per day. An increasedtolerance of subcutaneous dosing is anticipated as a result of adecreased Cmax compared with the same dose level administeredintravenously and more sustained levels of the rIL-15 product incirculation. Lowering the Cmax allows for more drug delivery overall.

To extend these findings to ALT-803, preclinical studies were conductedto evaluate i.v. (intravenous) and s.c. (subcutaneous) administration ofALT-803 in terms of the pharmacokinetics, immunostimulation andantitumor efficacy in C57BL/6 mice. Initial studies of C57BL/6 micetreated with 0.2 mg/kg ALT-801 showed an estimated half-life of 5.3hours for i.v. administration and 3.8 hours for s.c. administration. Themaximal serum concentration of ALT-803 was 650 ng/ml at 20 hour timepoint following s.c. administration and 1700 ng/ml at 2 hour time pointfollowing i.v. administration. In terms of immune stimulation, ALT-803administered s.c. or i.v. could equally induce proliferation of CD8+ Tcells and NK cells. Additionally, i.v. and s.c. administration ofALT-803 similarly activated immune cells to reduce tumor burden in thebone marrow of 5T33 myeloma-bearing mice. Both i.v. and s.c.administration of ALT-803 at up to 0.2 mg/kg was well tolerated innormal and tumor-bearing C57BL/6 mice.

A follow-up study for toxicological effects of 1 mg/kg ALT-803 injecteds.c. weekly for 4 weeks in C57BL/6 mice revealed immune system-relatedchanges that were similar to those seen in a previous toxicology studyin which the C57BL6 mice were treated with the same ALT-803 dosingregimen using an i.v. route. No mortalities were observed in micefollowing 4 weekly s.c. injections of 1 mg/kg ALT-803. With theexception of slight weight loss and the observance of hunched postureafter the first s.c. injection of ALT-803, no clinical signs of testarticle related toxicities were observed during this study. Examinationof the peripheral blood revealed that there were increased numbers ofWBC and lymphocyte counts compared to PBS controls. Overall, there was a9-fold increase for both WBCs and lymphocytes in animals treated withALT-803 compared to PBS injected mice. An increase in neutrophils,monocytes, eosinophils and basophils was also observed in s.c. ALT-803treated mice. Significant increases in the weight of spleen, lymph node,and liver (5.5, 3, and 1.3-fold respectively) of ALT-803 treated micewere observed. Comparable broad-based expansion of immune cells andincreased weights of lymphoid organs was previously reported for micereceiving multidose i.v. treatment with ALT-803.

Overall, the results of the preclinical studies of ALT-803 indicatedthat s.c. dosing decreases the Cmax compared to i.v. dosing, but retainsthe immunostimulatory activity and antitumor efficacy withoutexaggerating toxicity.

Example 8: Antitumor Activity of ALT-803 in Combination with ImmuneCheckpoint Blockers in Mice Bearing Orthotopic Bladder Tumors

In addition to the studies described in Example 4, the antitumoractivity of ALT-803 in combination with immune checkpoint blockers wasevaluated in mice bearing orthotopic MB49luc bladder tumors. C57BL/6mice (n=6/group) were instilled intravesically with MB49luc cells (3×10⁴cells/bladder) on study day 0, following polylysine pretreatment of thebladders. PBS, ALT-803 (0.2 mg/kg, i.v.) or ALT-803 (0.2 mg/kg) plusanti-PD-L1 and anti-CTLA4 Abs (each at 100 μg/injection, i.p.) wasadministered on 7, 10, 14, and 17 days post MB49luc tumor cellinstillation. The mice were maintained to assess survival rate among thetreatment groups as the efficacy endpoint. ALT-803 treatmentsignificantly prolonged the survival of the MB49luc bearing micecompared with PBS (FIG. 19A). However, the combination of ALT-803 withanti-PD-L1 and anti-CTLA4 Abs further prolonged survival compared tocontrol of monotherapy. This effect was also seen with combinationtherapy of ALT-803+anti-PD1 and ALT-803+anti-PD1/anti-CTLA4 mAbs (FIG.19B).

Additionally, mice that were cured of tumors by ALT-803 plusanti-PD-L1/anti-CTLA4 Ab therapy were resistant from bladder tumorrechallenge without further drug treatment whereas age-matchedtreatment-naïve mice developed tumors and die following tumor cellinstillation (FIG. 19A). These results indicate that ALT-803 monotherapyand combination therapy with anti-PD-L1, anti-PD-1 and anti-CTLA4 Abswas effective at treating bladder tumor bearing mice, including curativeresponses, and that the ALT-803/anti-PD-L1/anti-CTLA4 Ab combinationtherapy provided immune memory responses to subsequent tumor challenge.It is also noteworthy that the combination of ALT-803 plus checkpointinhibitor blockade did not cause any significant signs of toxicity inthe tumor-bearing animals for studies described above, which indicatesthat these combinations are well tolerated. As shown in FIG. 20 ,MB49luc cells express ligands for CTLA4 and PD-1. As such, these resultsare consistent with the antitumor activities of anti-CTLA4, anti-PD-1,and anti-PD-L1 Abs in combination ALT-803 in this tumor model.

Example 9: Combined Therapy of ALT-803 and Anti-Gp75 Antibody. TA99. InMurine Melanoma Model

The subcutaneous B16F10 melanoma tumor model in syngeneic C57BL/6 micewas used to further evaluate the efficacy of ALT-803 plus a therapeutictumor antigen specific antibody against solid tumors. This model alsohas the advantage of assessing the activity of T cells and other immunecells against established tumors and tumor rechallenge. One importantmelanoma-specific antigen for targeted therapy is gp75 (TYRP-1,tyrosinase-related protein-1), a 75 kDa protein involved in melaninsynthesis in melanosomes (Kobayashi T, et al. 1994. EMBO J. 13:5818-25).TA99 (mouse IgG2a) is a monoclonal antibody (mAb) specific for human andmurine gp75 (Thomson T M, et al. 1985. J Invest Dermatol. 85:169-74).Treatment with this antibody effectively abrogates subcutaneous murineB16F10 melanoma in syngeneic mice through the activation ofantibody-dependent cellular toxicity (ADCC) (Hara I, et al. 1995. J ExpMed. 182:1609-14).

The experiments described herein were designed to determine whether thisactivity could be further augmented by combination with ALT-803 andassess immune responses responsible for anti-tumor efficacy. In general,C57BL/6NHsd mice (7-week-old females) were injected s.c. on the lowerdorsal flank with 2×10⁵ B16F10 tumor cells in 200 μL PBS on study day 0(SD0). For all experiments in this study except tumor rechallenge,treatments were administered twice a week for two weeks (on study day10, 14, 17, 21 and 24) starting from SD10, the time point when more than75% of the animals had palpable B16F10 tumors. More specifically, micewere split into groups and injected with 200 μL PBS (i.v.) (vehiclecontrol), 0.2 mg/kg ALT-803 (i.v.), 10 mg/kg TA99 (i.v.), 100 μg/mouseanti-PD-L1 mAb 10F.9G2 (i.p.), or combination therapy of ALT-803, TA99and/or 10F.9G2. For depletion experiments, 200 μg/mouse depletionantibodies (anti-CD4 GK1.5, anti-CD8a 53.6.72 and anti-NK1.1 PK136) wereinjected i.p. once every week or 100 μL/mouse Clophosome(clodronate-loaded liposomes for macrophage depletion) were injectedi.p. once every 4 days, starting from SD3 and SD9 respectively, untilthe endpoint of the experiment. For experiments involving tumorrechallenge, 10 mg/kg TA99 (i.v.) was administered three times a week,0.2 mg/kg ALT-803 (i.v.) was administered once a week, for three weeksstarting from SD0. After about three months, tumor-free mice rescuedfrom the initial tumor challenge were injected s.c. contralaterally with2×10⁵ B16F10 tumor cells in 200 μL PBS. Tumor volumes were measureddaily starting from the first day of treatment to the end, andcalculated using formula ½(Length×Width²). Mice bearing a tumor loadwith one dimension>20 mm were sacrificed and counted as dead. Mice withno palpable tumor or a stable s.c. mass<50 mm³ were counted astumor-free.

In the initial study, mice (n=8) bearing established tumor were treatedwith TA99, ALT-803, TA99+ALT-803, or PBS control twice a week for twoweeks (FIG. 21A). Due to delayed therapeutic intervention, no tumorregression was observed in any of the treatment groups. However, tumorprogression was significantly inhibited by TA99 (p<0.001) and ALT-803(p<0.001) compared to PBS-treated group. Combined therapy resulted in asignificantly enhanced inhibition of tumor growth compared to eithermonotherapy (p<0.001), suggesting ALT-803 offers additional protectionto the antibody-dependent immunity against tumor. To determine whichimmune cell subsets are responsible for the ALT-803/TA99-mediatedanti-melanoma immunity, T lymphocyte, NK cells and macrophages weredepleted before and throughout the treatment course by intraperitonealadministration of cell type-specific antibodies or liposomes. Depletionof CD8⁺ T cells, NK cells and macrophages significantly loweredinhibition of tumor growth by the combined therapy (p<0.001; FIG. 21B),suggesting all three cell subsets contribute to the efficacy of ALT-803and TA99. Surprisingly, depletion of CD4⁺ T cells not only causedsignificant enhancement of tumor inhibition (p<0.001; FIG. 21B), butalso significant increase of animal survival (p<0.01; FIG. 21C) comparedto ALT-803+TA99 treatment group without depletion. Considering CD4⁺ Tcells are heavily involved in immune regulatory functions, this findingsuggest these cells (or a subset of CD4⁺ T cells) are immunosuppressiverather than immunoreactive in the initial ALT-803/TA99-mediated immunityagainst B16F10 tumors.

The effects of treatment on immune cells were examined in splenocytesand tumor-infiltrating leukocytes (TILs) harvested from mice bearingB16F10 melanoma tumors 3 days post-dosing therapy. Following ALT-803administration, there was an increase in the CD8⁺ T cell and NK cellpopulations, as well as a decrease in the CD4⁺ T cell, B cell andmacrophage populations, compared to the PBS control (FIG. 22A and FIG.22B). As expected, TA99 did not alter the percentages of immune cellsubsets in either splenocytes or TILs, except for causing a smallreduction of tumor-associated macrophages. The ALT-803/TA99 combinationshowed similar changes in the immune cell populations as ALT-803 alone.ALT-803 alone or combined with TA99 led to significant increase in thememory phenotype (CD44^(high)) of CD8⁺ T cells, supported by data fromboth splenocytes and TILs (FIG. 22C and FIG. 22D). Since the baselineCD8+CD44^(high) T cell population in TIL is two-fold greater than thiscell population in spleen, ALT-803-mediated expansion of memory CD8⁺ Tcell was less prominent in TILs (1.2-fold, p<0.01 vs. 2.5-fold inspleen, p<0.001). TA99 slightly reduced the percentage of memory CD8⁺ Tcell (p<0.05) in TILs, probably because more effector CD8⁺ T cells weredriven to participate in the antibody-dependent immunity againstmelanoma.

To assess whether the ALT-803-enhanced immune memory could contributedto improved long-term immunity against tumor cells, ALT-803+TA99treatment was started on study day 0 in an attempt to cure micechallenged with B16F10 tumor. In this treatment regimen, the addition ofALT-803 to TA99 therapy provided no improvement in animal survivalexcept that a moderate increase in the percentage of tumor-free animalsat SD60 was observed (FIG. 23A). However, when the surviving mice fromthe ALT-803+TA99 group were rechallenged with the B16F10 cells, asignificant delay in tumor development was observed when compared to theage-match treatment naïve mice (FIG. 23B). These findings support thehypothesis that previous treatment with ALT-803 induced immune cellresponses that are critical for the “vaccinal” protection of animalssubjected to subsequent tumor rechallenge. To assess which cells wereinvolved in this protective effect, depletion studies were conducted inALT-803+TA99 “cured” mice prior to tumor cell rechallenge. The resultsshowed that the ALT-803+TA99 “cured” mice administered PBS remainedprotected from B16F10 tumor rechallenge whereas those “cured” micetreated with antibodies depleting CD8, CD4 or NK cells showed no immuneprotection against mortality from subcutaneous B16F10 tumor rechallenge(FIG. 23C). These results indicate that all of these cell types areinvolved in the protective effects mediated by ALT-803+TA99 treatmentfollowing the initial tumor inoculation.

The role of immune cell activation and the PD-1/PD-L1 axis were alsoevaluated in this model. A single dose of ALT-803 upregulated CD25⁺cells in tumor-infiltrating CD4⁺ T cells (p<0.05), while TA99downregulated this activation marker (p<0.01; FIG. 24A). Moreover,ALT-803 treatment significantly increased PD-L1 expression on CD4⁺ Tcells both in periphery (p<0.001; FIG. 24B) and in the tumor (p<0.01;FIG. 24C). TA99 treatment resulted in a slight reduction of PD-L1expression on CD4⁺ T cells only in TIL (p<0.05), and TA99 with ALT-803was not sufficient to change the impact from ALT-803 alone. Hence, theeffectiveness of ALT-803 and its combination with therapeutic antibodiescould possibly be limited by the strengthened immune checkpointinhibitor pathway by CD4⁺ T cells.

ALT-803 effects on PD-1 expression on CD8⁺ T cells were also assessed inB16F10 tumor bearing mice. Single dose of ALT-803 led to moderatereduction of PD-1 expression on spleen CD8⁺ T cells compared to PBScontrol, restoring it to a level close to naive mice (FIG. 24C). Inaddition, ALT-803, TA99 and ALT-803+TA99 combination therapy all reducedPD-1 expression on tumor-infiltrating CD8⁺ T cells by about three fold(FIG. 24D).

One important anti-tumor mechanism of action of IL-15 is through theactivation of NK cells. When the activation marker KLRG1 was examined onNK cells, it was found that B16F10 melanoma, though poorly immunogenic,can induce a moderate increase of KLRG1 on spleen NK cells (p<0.05; FIG.25A). ALT-803 treatment further increased the activation of NK cells inspleen (p<0.05; FIG. 25A) and more importantly, it caused a largeincrease of KLRG1 expression on tumor-infiltrating NK cells (4-fold,p<0.001; FIG. 25B). This might explain why ALT-803 can effectively boostTA99-mediated ADCC against B16F10 tumors where NK cells are a majorimmune cell involved in this response. Like CD8⁺ T cells, ALT-803 alsodownregulated PD-1 expression on NK cells in spleen (p<0.01; FIG. 25C)and tumor (p<0.05; FIG. 25D), implying that ALT-803 could intrinsicallyattenuate the inhibition from checkpoint pathway through the PD-1 arm ofCD8⁺ T cells and NK cells, independent of additional checkpointblockade.

In order to explore whether checkpoint blockade would further enhancethe combinatorial anti-tumor function of ALT-803 and TA99, mice (n=8)bearing established B16F10 tumors were treated with ALT-803+TA99 withand without anti-PD-L1 mAb 10F.9G2 in the delayed intervention model(FIG. 26A). Anti-PD-L1 mAb alone slowed tumor growth by a small factor(p<0.05). Thus, despite the observation that B16F10 cells constitutivelyexpress PD-L1 both in vitro and in vivo (FIG. 26B), anti-PD-L1 mAbfailed to be a potent monotherapy for the inhibition of melanoma growthin this model. Judging from the tumor growth curve, ALT-803+TA99combined with anti-PD-L1 mAb did exhibit greater anti-tumor activitywhen compared to ALT-803+TA99 therapy (p<0.05).

Overall, the results of these studies indicate that ALT-803 incombination with a tumor-specific antibody can provide antitumoractivity and prolong survival of mice bearing established solid tumors.This treatment also provides immune protection against further tumorrechallenge. These effects are mediated by NK and T cells which areactivated and proliferate in response to treatment. Additionally,therapeutic combinations with checkpoint inhibitors can further augmentthe antitumor efficacy of the ALT-803+tumor-specific antibody regimen.As a result, ALT-803 monotherapy and ALT-803 combination therapeuticstrategies are applicable to various treatment approaches for neoplasia,including adjuvant, neo-adjuvant, induction, consolidation, maintenance,first line, ≥second line treatment and combinations with surgery,radiation or chemotherapy.

It is also noteworthy that ALT-803 alone or in combination withanti-tumor antibodies and/or checkpoint inhibitor blockade did not causeany significant signs of toxicity in the tumor-bearing animals forstudies described above, which indicates that these combinations arewell tolerated.

Example 10: Clinical Studies of ALT-803 in Patients with Malignancies

ALT-803 is being evaluated in patients with malignancies as follows.

A multi-center clinical study of ALT-803 is underway in patients withmetastatic melanoma and other solid tumors. The study is being conductedas a dose escalation with one patient each to be enrolled in the firsttwo cohorts and a minimum of three patients to be enrolled in the lastthree cohorts to determine the maximum tolerated dose (MTD) or OptimumBiological Dose (OBD) of ALT-803. Enrolled patients receive two 6-weekcycles consisting of 4 weekly ALT-803 intravenous doses followed by a2-week rest period. Patients with stable or benefitting disease will beeligible to receive up to two additional 6-week cycles. One patientenrolled to the 0.3 μg/kg ALT-803 dose level. The reported studydrug-attributed adverse events were transient low-grade fever, rigors,nausea and vomiting. The next patient enrolled to the 0.5 μg/kg doselevel. All reported adverse events were mild to moderate includingnausea, fatigue and pruritus. Three patients enrolled and completed thestudy treatment at the 1 μg/kg ALT-803 dose level. All adverse eventsreported for these patients were mild to moderate, including chills,worsening constipation and hypertension. The study protocol for thistrial was amended to include renal cell carcinoma, non-small cell lungcarcinoma and squamous cell head and neck carcinoma. Three patients,including one patient with renal cell carcinoma, have enrolled to the 3μg/kg dose level. Adverse events reported so far were mild to moderateincluding fever, fatigue, vomiting and myalgia. There have been no doselimiting toxicities, Grade 3/4 toxicities or severe adverse events inany of these ALT-903 treated patients, which indicates that thistreatment was well tolerated. Disease stabilization has been reported insome patients and treatment-mediated clinical benefit (includingdecreased tumor burden, disease progression or relapse or toxicity, orincreased progression free survival, time to progression, duration ofresponse, survival, or quality of life) is expected.

A multi-center clinical study of ALT-803 is underway in patients withhematologic malignancy who have relapsed after autologous stem celltransplantation (ASCT). The first phase of this study is being conductedunder a standard 3+3 design of dose escalation for toxicity. Enrolledpatients receive ALT-803 intravenous doses given once weekly for 4weeks. Six patients have enrolled and completed the study treatment atthe 1 μg/kg ALT-803 dose level. For the first three patients, thereported study drug-attributed adverse events were fever, chills, rigorsand edema. Grade 1 fever in two patients occurred approximately 4 to 5hours after ALT-803 dosing and then subsided approximately 6 to 7 hoursafter ALT-803 dosing. Grade 2 rigors occurred in two patients, grade 2chills occurred in two patients and grade 1 chills occurred in onepatient. Grade 2 rigors in one patient required Demerol for 3 out of 4ALT-803 doses. One patient experienced grade 2 edema and another patientexperienced grade 1 edema. The first three patients experiencedasymptomatic hypotension, but the patients were normotensive after fluidadministration without the recurrence of hypotension episodes. None ofthe treated patients were pre-hydrated with fluids. Grade 2 skin rashwas also observed in one patient after the second dose of ALT-803, whichwas consistent with graft-versus-host disease. The fourth, fifth andsixth patients received the study treatment with no reported AEs. Threepatients completed the study treatment at the 3 μg/kg ALT-803 doselevel. The patients received hydration prior to each dose, reportedadverse events include grade 1 fever and chills 6 and 10 hours afterALT-803 dosing. Three patients enrolled and completed treatment at the 6μg/kg ALT-803 dose level. Most common reported adverse events in thiscohort include mild to moderate fever, rigors and flu-like symptoms. Twopatients are being treated at the 10 μg/kg ALT-803 dose level. Reportedadverse events for the first patient after the first dose of ALT-803include transient fever, nausea and vomiting. The adverse events startedaround 3 hours after dosing and lasted approximately 4 hours. Thepatient also experienced low grade asymptomatic hypotension. IV fluidswere administered and the blood pressure returned to baseline. Thesecond patient experienced transient fever and chills after the firstdose of ALT-803. The chills were controlled with Demoral. There havebeen no dose limiting toxicities, Grade 3/4 toxicities or severe adverseevents in any of these ALT-903 treated patients, which indicates thatthis treatment was well tolerated. The protocol was amended to changethe administration of ALT-803 from IV to subcutaneous injection startingat the 6 μg/kg dose level. Patient enrollment will continue at the 10μg/kg dose level with i.v. administration until a total of threepatients are enrolled in this cohort. Patient enrollment forsubcutaneous injection at 6 μg/kg will then be initiated.Treatment-mediated clinical benefit (including decreased tumor burden,disease progression or relapse or toxicity, or increased progressionfree survival, time to progression, duration of response, survival, orquality of life) is expected.

Clinical biomarker assessment is being conducted. For the study ofpatients with hematologic malignancy after ASCT, preliminary data isavailable on the Ki-67 analysis of NK, CD4⁺, CD8⁺ and NKT cell subsetsand serum cytokines of the patients' pre-dose and post-dose specimens.Serum levels of both IFN-γ and IL-6 were induced in a dose-dependentmanner within the dose range from 1 μg/kg to 6 μg/kg ALT-803. Ki67⁺ NK,CD8⁺ and CD4⁺ T cells increased after ALT-803 dosing at a dose level of≥3 μg/kg in all patients. Thus, the preliminary data suggests thatALT-803 consistently promotes the activation and proliferation of NK, Tand NKT cells for patients at a dose level of ≥3 μg/kg with thisindication. Similarly, serum levels of IFN-γ and IL-6 were induced inpatients with solid tumors following administration of ALT-803,indicating treatment-related immune stimulation in these patients.

A multi-center clinical study of ALT-803 is underway in patients withrelapsed or refractory multiple myeloma. The first phase of this studyincludes a classic (3+3) dose escalation to determine the MTD or minimumefficacious dose (MED) and to designate a dose level for the phase IItwo-stage expansion. The dose levels are 1, 3, 6, 10 and 20 μg/kg ofALT-803. Enrolled patients will receive two 6-week cycles consisting of4 weekly ALT-803 intravenous doses followed by a 2-week rest period.Patients with stable or benefitting disease will be eligible to receiveup to two additional 6-week cycles. Three patients enrolled andcompleted the study treatment at the 1 μg/kg ALT-803 dose level. Alladverse events reported for these patients were mild to moderate,including constipation, nausea, fatigue, ALC decreased and WBC countdecreased. All patients are receiving pre-medications. Two patients areundergoing treatment at the 3 μg/kg ALT-803 dose level. Reported adverseevents include mild to moderate fevers, rigors and neutropenia. Therehave been no dose limiting toxicities, Grade 3/4 toxicities or severeadverse events in any of these ALT-903 treated patients, which indicatesthat this treatment was well tolerated. Treatment-mediated clinicalbenefit (including decreased tumor burden, disease progression orrelapse or toxicity, or increased progression free survival, time toprogression, duration of response, survival, or quality of life) isexpected. Serum levels of IFN-γ and IL-6 were induced in patients withmultiple myeloma following administration of ALT-803, indicatingtreatment-related immune stimulation in these patients.

A multi-center clinical study of ALT-803 in combination with BacillusCalmette-Guerin (BCG) is in patients with BCG-naïve non-muscle invasivebladder cancer. The first phase of this study includes a classic (3+3)dose escalation to determine the MTD of ALT-803 and to determine therecommended dose (RD) of ALT-803 combined with BCG for the expansionphase. The dose levels are 100, 200 and 400 μg/instillation of ALT-803plus standard BCG (50 mg/instillation). The expansion phase consists ofa noncomparative randomized design of patients receiving ALT-803 at theRD level in combination with BCG or BCG alone. Enrolled patients willreceive BCG plus ALT-803 weekly via a urinary catheter in the bladderfor 6 consecutive weeks. Three patients enrolled and completed treatmentin the first cohort of 100 μg/instillation of ALT-803 plus BCG. Reportedstudy drug-attributed adverse events included mild nausea, headache,hematuria and urinary tract pain and moderate cystitis noninfective.Three patients have enrolled and completed treatment in the 200μg/instillation of ALT-803 plus BCG. Reported study drug-attributedadverse events included mild hematuria and urinary incontinence. Twopatients enrolled are ongoing treatment in the 400 μg/instillation ofALT-803 cohort. There have been no dose limiting toxicities, Grade 3/4toxicities or severe adverse events in any of these ALT-903 plus BCGtreated patients, which indicates that this treatment was welltolerated. A number of these treated patients exhibited no diseaserecurrence (considered a complete response in this indication) for atleast 9 months post therapy, suggesting treatment-related clinicalactivity. Treatment-related increases in urinary cytokines were alsoobserved in some patients. Treatment-mediated clinical benefit(including decreased tumor burden, disease progression or relapse ortoxicity, or increased progression free survival, time to progression,duration of response, survival, or quality of life) is expected.

A multi-center clinical study of ALT-803 plus rituximab is underway inpatients with relapsed or refractory indolent B cell non-Hodgkinlymphoma. The first phase of this study includes a classic (3+3) doseescalation to determine the MTD or MED and to designate a dose level forthe phase II two-stage expansion. The dose levels are 1, 3 and 6 μg/kgof ALT-803. Enrolled patients will receive a 4-week induction cycleconsisting of 4 weekly doses of ALT-803 and standard rituximab (375mg/m2) by intravenous injection. Patients with stable or benefittingdisease will be eligible to receive up to four consolidation treatmentcycles consisting of a single treatment of ALT-803 plus rituximab,repeated every 8 weeks for a total of 4 additional ALT-803 and rituximabdoses. One patient enrolled and is currently undergoing treatment at the1 μg/kg ALT-803 dose level. Adverse events reported thus far for thispatient were mild to moderate including edema, ALC decreased and WBCcount decreased. There have been no dose limiting toxicities, Grade 3/4toxicities or severe adverse events in the ALT-903+rituximab treatedpatient, which indicates that this treatment was well tolerated.Treatment-mediated clinical benefit (including decreased tumor burden,disease progression or relapse or toxicity, or increased progressionfree survival, time to progression, duration of response, survival, orquality of life) is expected.

A multi-center clinical study of ALT-803 plus nivolumab (anti-PD-1 Ab)will be conducted in patients with advanced or metastatic non-small celllung cancer. The first phase of this study includes a dose escalation todetermine the MTD of ALT-803 and to designate a dose level for the phaseII two-stage expansion. The dose levels are 6, 10, and 15 μg/kg ofsubcutaneous ALT-803. Enrolled patients will receive two 6-week cyclesconsisting of 5 weekly doses of ALT-803 and standard intravenouslynivolumab every 2 weeks (3 mg/kg). Patients with stable or benefittingdisease will be eligible to receive additional 6-week ALT-803 plusnivolumab cycles. Treatment-mediated clinical benefit (includingdecreased tumor burden, disease progression or relapse or toxicity, orincreased progression free survival, time to progression, duration ofresponse, survival, or quality of life) is expected.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of treating non-muscle invasive bladdercancer, the method comprising: administering by instillation into thebladder of a human subject in need thereof a pharmaceutical compositioncomprising i) an effective amount of Bacillus Calmette-Guerin (BCG) andii) an effective amount of an Interleukin-15/Interleukin-15 Receptoralpha sushi (IL-15:IL-15RαSu) complex, wherein the IL-15 comprises awild type IL-15.
 2. The method of claim 1, wherein the subject hasBCG-naive non-muscle invasive bladder cancer.
 3. The method of claim 1,wherein at least one binding partner of the IL-15:IL-15RαSu complex is afusion protein, wherein the at least one binding partner is bound to abiologically active polypeptide.
 4. The method of claim 3, wherein theIL-15RαSu is genetically fused to an immunoglobulin Fc domain, orfunctional fragment thereof, wherein an IL-15RαSuFc fusion protein isformed.
 5. The method of claim 4 wherein the IL-15RαSuFc fusion proteinnoncovalently binds IL-15 to form a fusion protein complex.
 6. Themethod of claim 5 wherein the IL-15/IL-15RαSuFc complex comprises adimeric IL-15RαSu and two IL-15 molecules.